Method for controlled dna fragmentation

ABSTRACT

A composition and method for controlled in vitro fragmentation of nucleic acids. A transposase forms catalytically active complexes with a modified transposon end that contains within its end sequence degenerate, apurinic/apyrimidinic sites, nicks, or nucleotide gaps, to fragment or shear a target nucleic acid sample in a controlled process. This method yields desired average nucleic acid fragment sizes. The inventive composition and method may be applied for generation of DNA fragments containing shortened transposon end sequences to facilitate subsequent reactions, for production of asymmetrically tailed DNA fragments, etc.

This application claims priority to U.S. Provisional Application No.61/934,879, filed on Feb. 3, 2014.

Throughout this application various publications, patents, and/or patentapplications are referenced. The disclosures of these publications,patents, and/or patent applications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF INVENTION

The invention relates to the field of controlled fragmentation ofnucleic acids.

SUMMARY

In some embodiments, the disclosure relates generally to compositions,as well as related methods, systems, kits and apparatuses, comprising atranspososome complex.

In some embodiments, a transpososome complex comprises: (i) a pluralityof transposases, (ii) a polynucleotide containing a first transposon endsequence, and (iii) a polynucleotide containing a second transposon endsequence.

In some embodiments, the first transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the second transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the first transposon end sequence contains at leastone modification, including a lesion such as a nick, gap, apurinic siteor apyrimidinic site.

In some embodiments, the second transposon end sequence contains atleast one modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequencescontain at least one modification, including a lesion such as a nick,gap, apurinic site or apyrimidinic site.

In some embodiments, the first or the second transposon end sequencelacks a modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments, a transpososome complex comprises: (i) a pluralityof transposases, (ii) a polynucleotide containing a first transposon endsequence, wherein the first transposon end sequence contains at leastone modification, including a lesion such as a nick, gap, apurinic siteor apyrimidinic site, and (iii) a polynucleotide containing a secondtransposon end sequence, wherein the second transposon end sequencecontains at least one modification, including a lesion such as a nick,gap, apurinic site or apyrimidinic site. In some embodiments, the firsttransposon end sequence is capable of binding to the plurality oftransposases. In some embodiments, the second transposon end sequence iscapable of binding to the plurality of transposases. Optionally, thetranspososome complex comprises two, three, four or more transposases.

In some embodiments, the transpososome complex is contained in a singlereaction mixture. For example, the single reaction mixture can becontained in a single reaction vessel or in a single well.

In some embodiments, the transpososome complex can be produced byconducting any method for preparing the transposon complex, or anymethod for fragmenting DNA in vitro, described herein.

In some embodiments, the disclosure relates generally to compositions,as well as related methods, systems, kits and apparatuses, comprising atranspososome/target nucleic acid complex.

In some embodiments a transpososome/target nucleic acid complexcomprises: (i) a plurality of transposases, (ii) a polynucleotidecontaining a first transposon end sequence, (iii) a polynucleotidecontaining a second transposon end sequence, and (iv) a target nucleicacid molecule. In some embodiments, the target nucleic acid moleculecomprises a target DNA molecule.

In some embodiments, the first transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the second transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the first transposon end sequence contains at leastone modification, including a lesion such as a nick, gap, apurinic siteor apyrimidinic site.

In some embodiments, the second transposon end sequence contains atleast one modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequencescontain at least one modification, including a lesion such as a nick,gap, apurinic site or apyrimidinic site.

In some embodiments, the first or the second transposon end sequencelacks a modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments a transpososome/target nucleic acid complexcomprises: (i) a plurality of transposases, (ii) a polynucleotidecontaining a first transposon end sequence, wherein the first transposonend sequence is capable of binding to the plurality of transposases andwherein the first transposon end sequence contains at least onemodification, including a lesion such as a nick, gap, apurinic site orapyrimidinic site, (iii) a polynucleotide containing a second transposonend sequence, wherein the second transposon end sequence is capable ofbinding to the plurality of transposases and wherein the secondtransposon end sequence contains at least one modification, including alesion such as a nick, gap, apurinic site or apyrimidinic site, and (iv)a target nucleic acid molecule. In some embodiments, the target nucleicacid molecule comprises a target DNA molecule. Optionally, thetranspososome complex comprises two, three, four or more transposases.

In some embodiments, the transpososome/target DNA complex can beproduced by conducting any method for preparing the transposome/targetDNA complex, or any method for fragmenting DNA in vitro, describedherein.

In some embodiments, the disclosure relates generally to compositions,as well as related methods, systems, kits and apparatuses, comprising anucleic acid fragmentation reaction mixture.

In some embodiments, the nucleic acid fragmentation reaction mixturecomprises: (i) a plurality of transposases, (ii) a polynucleotidecontaining a first transposon end sequence, (iii) a polynucleotidecontaining a second transposon end sequence, (iv) a target nucleic acidmolecule, and (v) an activating cation.

In some embodiments, the first transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the second transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the first transposon end sequence contains at leastone modification, including a lesion such as a nick, gap, apurinic siteor apyrimidinic site.

In some embodiments, the second transposon end sequence contains atleast one modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequencescontain at least one modification, including a lesion such as a nick,gap, apurinic site or apyrimidinic site.

In some embodiments, the first or the second transposon end sequencelacks a modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments, the activating agent includes one or anycombination of magnesium and/or manganese.

In some embodiments, the nucleic acid fragmentation reaction mixturecomprises: (i) a plurality of transposases, (ii) a polynucleotidecontaining a first transposon end sequence, wherein the first transposonend sequence is capable of binding to the plurality of transposases andwherein the first transposon end sequence contains at least onemodification, including a lesion such as a nick, gap, apurinic site orapyrimidinic site, (iii) a polynucleotide containing a second transposonend sequence, wherein the second transposon end sequence is capable ofbinding to the plurality of transposases and wherein the secondtransposon end sequence contains at least one modification, including alesion such as a nick, gap, apurinic site or apyrimidinic site, (iv) atarget nucleic acid molecule, and (v) an activating cation (e.g.,magnesium or manganese). In some embodiments, the target nucleic acidmolecule comprises a target DNA molecule.

In some embodiments, the nucleic acid fragmentation reaction mixturefurther comprises a buffer (e.g., Tris-HCl), an alkali metal (e.g., NaCland/or KCl), a detergent (e.g., TritonX-100, TritonX-114, NP-40, Brij,Tween-20, SDS, or CHAPS), and an activating cation (e.g., magnesium ormanganese). Optionally, the nucleic acid fragmentation reaction mixturelacks any activating cation (e.g., magnesium or manganese). For example,an activating cation includes any cation required by a transposase forcatalyzing a transposition reaction. In some embodiments, a nucleic acidfragmentation reaction mixture lacks an activating cation (or contains avery low level of activating cation).

In some embodiments, the disclosure relates generally to compositions,as well as related methods, systems, kits and apparatuses, comprising afragmented nucleic acid molecule.

In some embodiments, the fragmented nucleic acid molecule comprises: (i)a first end of the DNA molecule joined to the first transposon endsequence and (ii) a second end of the DNA molecule joined to the secondtransposon end sequence.

In some embodiments, the first transposon end sequence includes at leastone modification, including a lesion such as a nick, gap, apurinic siteor apyrimidinic site.

In some embodiments, the second transposon end sequence includes atleast one modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequencescontain at least one modification, including a lesion such as a nick,gap, apurinic site or apyrimidinic site.

In some embodiments, the first or the second transposon end sequencelacks a modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments, the fragmented DNA molecule, or a plurality offragmented DNA molecules, can be produced by conducting any method forpreparing a plurality of transposon complexes, or any method forfragmenting DNA in vitro, described herein.

In some embodiments, the plurality of fragmented DNA molecules have asize range of about 100-2000 bp, or about 100-250 bp, or about 250-500bp, or about 500-750 bp, or about 750-1000 bp, or about 1000-1250 bp, orabout 1250-1500 bp, or about 1500-1750 bp, or about 1750-2000 bp.

In some embodiments, the disclosure relates generally to compositions,as well as related methods, systems, kits and apparatuses, comprising asingle reaction mixture containing a nucleic acid amplification reactionmixture.

In some embodiments, the amplification step is streamlined by conductingthe amplification reaction in the same reaction mixture, and in the samereaction vessel, that is used to conduct the nucleic acid fragmentationstep without any intervening steps to remove the fragmentation reactionmixture. The reaction mixture used to fragment target DNA molecules,using any of the transpososome complexes or any of thetranspososome/target nucleic acid complexes described herein, can alsobe used to amplify the fragmented DNA molecules. Optionally, the nucleicacid fragmentation and the amplification steps are performed in adifferent reaction mixture, in the same or in a different reactionvessel.

In some embodiments, the single reaction mixture containing a nucleicacid amplification reaction mixture comprises: a nucleic acidfragmentation reaction mixture, at least one fragmented DNA molecule andat least one component for amplifying nucleic acids.

For example, components for amplifying nucleic acids include any one orany combination of: primers, polymerase and/or nucleotides.

In some embodiments, the single reaction mixture containing a nucleicacid amplification reaction mixture comprises: (i) a nucleic acidfragmentation reaction mixture, (ii) at least one fragmented DNAmolecule, (iii) one or more primers that hybridize with at least aportion of the fragmented DNA molecule or a sequence that iscomplementary to the fragmented DNA molecule, (iv) one or morepolymerases, and (v) one or more nucleotides.

For example, the nucleic acid fragmentation reaction mixture includesany one or any combination of: a buffer (e.g., Tris-HCl), a salt (e.g.,NaCl and/or KCl), a detergent (e.g., TritonX-100), and/or an activatingcation (e.g., magnesium or manganese).

In some embodiments, at least one end of a fragmented DNA molecule isjoined to the first transposon end sequence having at least onemodification. Optionally, the at least one modification includes alesion such as a nick, gap, apurinic site or apyrimidinic site.

In some embodiments, at least one end of a fragmented DNA molecule isjoined to the second transposon end sequence having at least onemodification. Optionally, the at least one modification includes alesion such as a nick, gap, apurinic site or apyrimidinic site.

In some embodiments, the fragmented DNA molecule is joined at a firstend to a first transposon end sequence, and is joined at a second end toa second transposon end sequence, and the first and the secondtransposon end sequences have identical or different sequences.

In some embodiments, the disclosure relates generally to compositions,as well as related methods, systems, kits and apparatuses, comprisingtranspososome complexes in the presence of one or more stabilizingagents.

In some embodiments, the transpososome complexes comprise: (i) aplurality of transposases, (ii) a polynucleotide containing a firsttransposon end sequence, (iii) a polynucleotide containing a secondtransposon end sequence, and (iv) at least one stabilizing agent.

In some embodiments, the first transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the second transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the first transposon end sequence contains at leastone modification, including a lesion such as a nick, gap, apurinic siteor apyrimidinic site.

In some embodiments, the second transposon end sequence contains atleast one modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequencescontain at least one modification, including a lesion such as a nick,gap, apurinic site or apyrimidinic site.

In some embodiments, the first or the second transposon end sequencelacks a modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments, the stabilizing agent includes any compound thatstabilizes the structure, conformation and/or activity of a protein orenzyme. In some embodiments, the stabilizing agent includes any compoundthat increases the solubility of a protein or enzyme in solution. Insome embodiments, the stabilizing agent includes any compound thatdecreases protein aggregation.

In some embodiments, one or more stabilizing agents is added before,during or after the transpososome complexes are formed. In someembodiments, the stabilizing agent includes any compound that, whenadded to the transpososome complexes, helps retain some or alltranspososome-mediated activity. For example, the presence of one ormore stabilizing agent can retain about 5-20%, or about 20-40%, or about40-60%, or about 60-80%, or about 80-95%, or about 95-100% activity.Optionally, the shelf-life of the transpososome complexes can beextended by adding one or more stabilizing agents. Optionally, thetranspososome complexes, in the presence of at least one stabilizingagent, can retain some or all enzyme activity during shipping. In someembodiments, the transpososome complexes, in the presence of at leastone stabilizing agent, can be stored or shipped at about −20° C.

In some embodiments, the transpososome complexes comprise: (i) aplurality of transposases, (ii) a polynucleotide containing a firsttransposon end sequence, wherein the first transposon end sequence iscapable of binding to the plurality of transposases and wherein thefirst transposon end sequence contains at least one modification,including a lesion such as a nick, gap, apurinic site or apyrimidinicsite, (iii) a polynucleotide containing a second transposon endsequence, wherein the second transposon end sequence is capable ofbinding to the plurality of transposases and wherein the secondtransposon end sequence contains at least one modification, including alesion such as a nick, gap, apurinic site or apyrimidinic site, and (iv)at least one stabilizing agent.

For example, the stabilizing agent includes any amino acid includingcharged amino acids. Optionally, the stabilizing agent includes any oneor any combination of arginine, histidine, lysine, aspartic acid,glutamic acid (Golovanvo, et al., 2004 Journal of Am. Chem. Soc.126(29):8933-8939, Baynes, Wang and Trout 2005 Biochemistry44(12):4919-4925, Shukla and Trout 2011 Journal of Phys. Chem B115(41):11831). In some embodiments, the stabilizing agent includes amixture of arginine and glutamic acid. In some embodiments, thestabilizing agent comprises a polyol. In some embodiments, thestabilizing agent includes any one or any combination of glycol,propylene glycol, and/or glycerol. In some embodiments, the stabilizingagent comprises a polysaccharide. In some embodiments, the stabilizingagent comprises any one or any combination of sucrose, trehalose,polyhydric alcohol, glucose (e.g., L- or D-glucose), and/or galactose(e.g., D-galactose). In some embodiments, the transpososome complexincludes BSA.

In some embodiments, the disclosure relates generally to kits, as wellas related compositions, methods, systems, and apparatuses, comprisingcomponents for assembling transpososome complexes, including: (i) aplurality of transposases, (ii) a polynucleotide containing a firsttransposon end sequence, and (iii) a polynucleotide containing a secondtransposon end sequence.

In some embodiments, the disclosure relates generally to kits, as wellas related compositions, methods, systems, and apparatuses, comprisingpre-assembled transpososome complexes, which include: (i) a plurality oftransposases, (ii) a plurality of polynucleotides containing a firsttransposon end sequence, and (iii) a plurality of polynucleotidescontaining a second transposon end sequence. Optionally, thepre-assembled transpososome complexes comprises two, three, four or moretransposases.

In some embodiments, the first transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the second transposon end sequence is capable ofbinding to the plurality of transposases.

In some embodiments, the first transposon end sequence contains at leastone modification, including a lesion such as a nick, gap, apurinic siteor apyrimidinic site.

In some embodiments, at least one first transposon end sequence lacks amodification (e.g., a lesion such as a nick, gap, apurinic site orapyrimidinic site).

In some embodiments, the plurality of first transposon end sequencesincludes a plurality of double-stranded polynucleotides having a firststrand (e.g., an attacking strand) and second strand (e.g., anon-attacking strand).

In some embodiments, the second transposon end sequence contains atleast one modification, including a lesion such as a nick, gap, apurinicsite or apyrimidinic site.

In some embodiments, at least one second transposon end sequence lacks amodification (e.g., a lesion such as a nick, gap, apurinic site orapyrimidinic site).

In some embodiments, the plurality of second transposon end sequencesincludes a plurality of double-stranded polynucleotides having a firststrand (e.g., an attacking strand) and second strand (e.g., anon-attacking strand).

In some embodiments, the first and the second transposon end sequencescontain at least one modification, including a lesion such as a nick,gap, apurinic site or apyrimidinic site.

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments, the kits, as well as related compositions, methods,systems, and apparatuses, further comprise one or more activatingcation. For example, the activating cation includes magnesium andmanganese.

In some embodiments, the kits, as well as related compositions, methods,systems, and apparatuses, further comprise one or more stabilizingagent. For example, the stabilizing agent includes any compound thatstabilizes the structure, conformation and/or activity of a protein orenzyme. In some embodiments, the stabilizing agent includes any compoundthat increases the solubility of a protein or enzyme in solution. Insome embodiments, the stabilizing agent includes any compound thatdecreases protein aggregation. For example, the stabilizing agentincludes any amino acid including charged amino acids. Optionally, thestabilizing agent includes any one or any combination of arginine,histidine, lysine, aspartic acid, glutamic acid (Golovanvo, et al., 2004Journal of Am. Chem. Soc. 126(29):8933-8939, Baynes, Wang and Trout 2005Biochemistry 44(12):4919-4925, Shukla and Trout 2011 Journal of Phys.Chem B 115(41):11831). In some embodiments, the stabilizing agentincludes a mixture of arginine and glutamic acid. In some embodiments,the stabilizing agent comprises a polyol. In some embodiments, thestabilizing agent includes any one or any combination of glycol,propylene glycol, and/or glycerol. In some embodiments, the stabilizingagent comprises a polysaccharide. In some embodiments, the stabilizingagent comprises any one or any combination of sucrose, trehalose,polyhydric alcohol, glucose (e.g., L- or D-glucose), and/or galactose(e.g., D-galactose). In some embodiments, the kits, as well as relatedcompositions, methods, systems, and apparatuses, further comprise BSA.

In some embodiments, the kits, as well as related compositions, methods,systems, and apparatuses, further comprise one or more containers forholding the components for assembling transpososome complexes or thepre-assembled transpososome complexes.

In some embodiments, the kits, as well as related compositions, methods,systems, and apparatuses, can also include buffers and reagents. Forexample, the buffers can include Tris, Tricine, HEPES, or MOPS, orchelating agents such as EDTA or EGTA. The buffers or reagents caninclude monovalent ions, such as KCl, K-acetate, NH₄-acetate,K-glutamate, NH₄Cl, or ammonium sulfate. In yet another example, thereagents can include divalent ions, such as Ca²⁺, Mg²⁺, Mn²⁺, or CaCl₂,MgCl₂, MnCl₂, or Mg-acetate, and the like.

In some embodiments, the kits, as well as related compositions, methods,systems, and apparatuses, can also include instructions for performingthe controlled nucleic acid fragmentation reactions.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes.

In some embodiments, methods for preparing a plurality of transpososomecomplexes comprise contacting a plurality of transposases with aplurality of polynucleotides.

In some embodiments, the plurality of transposases is contacted with theplurality of polynucleotides in a single reaction mixture. For example,the single reaction mixture can be contained in a single reaction vesselor a single well.

In some embodiments, methods for preparing a plurality of transpososomecomplexes comprise contacting in a single reaction mixture a pluralityof transposases with a plurality of polynucleotides.

In some embodiments, the plurality of polynucleotides contain aplurality of first transposon end sequences, a plurality of secondtransposon end sequences, or a mixture of first and second transposonend sequences.

In some embodiments, the first transposon end sequences are capable ofrecognizing and binding a transposase.

In some embodiments, the plurality of first transposon end sequencesincludes at least one modification. Optionally, the at least onemodification includes a lesion such as a nick, gap, apurinic site orapyrimidinic site.

In some embodiments, at least one first transposon end sequence lacks amodification (e.g., a lesion such as a nick, gap, apurinic site orapyrimidinic site).

In some embodiments, the plurality of first transposon end sequencesincludes a plurality of double-stranded polynucleotides having a firststrand (e.g., an attacking strand) and second strand (e.g., anon-attacking strand).

In some embodiments, the second transposon end sequences are capable ofrecognizing and binding a transposase.

In some embodiments, the plurality of second transposon end sequencesincludes at least one modification. Optionally, the at least onemodification includes a lesion such as a nick, gap, apurinic site orapyrimidinic site.

In some embodiments, at least one second transposon end sequence lacks amodification (e.g., a lesion such as a nick, gap, apurinic site orapyrimidinic site).

In some embodiments, the plurality of second transposon end sequencesincludes a plurality of double-stranded polynucleotides having a firststrand (e.g., an attacking strand) and second strand (e.g., anon-attacking strand).

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments, one or more stabilizing agents is added before,during or after the transpososome complexes are formed. For example, thestabilizing agent includes any amino acid including charged amino acids.Optionally, the stabilizing agent includes any one or any combination ofarginine, histidine, lysine, aspartic acid, glutamic acid (Golovanvo, etal., 2004 Journal of Am. Chem. Soc. 126(29):8933-8939, Baynes, Wang andTrout 2005 Biochemistry 44(12):4919-4925, Shukla and Trout 2011 Journalof Phys. Chem B 115(41):11831). In some embodiments, the stabilizingagent includes a mixture of arginine and glutamic acid. In someembodiments, the stabilizing agent comprises a polyol. In someembodiments, the stabilizing agent includes any one or any combinationof glycol, propylene glycol, and/or glycerol. In some embodiments, thestabilizing agent comprises a polysaccharide. In some embodiments, thestabilizing agent comprises any one or any combination of sucrose,trehalose, polyhydric alcohol, glucose (e.g., L- or D-glucose), and/orgalactose (e.g., D-galactose). In some embodiments, the transpososomecomplex includes BSA.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, comprise: (a)contacting in a single reaction mixture (i) a plurality of transposases,(ii) a plurality of polynucleotides containing a first transposon endsequence, wherein the first transposon end sequence is capable ofbinding to a transposase from the plurality of transposases, and (iii) aplurality of polynucleotides containing a second transposon endsequence, wherein the second transposon end sequence is capable ofbinding to a transposase from the plurality of transposases; and (b)forming at least one transpososome complex having a transposase, a firsttransposon end sequence, and a second transposon end sequence, whereinthe first transposon end sequence contains at least one modification,including a lesion such as at least one nick, gap, apurinic site orapyrimidinic site, and wherein the second transposon end sequenceoptionally contains at least one modification, including a lesion suchas at least one nick, gap, apurinic site or apyrimidinic site.Optionally, the transposome complex may contain a plurality oftransposases, including two, three, four or more transposases.Optionally, one or more stabilizing agents is added after thetranspososome complexes are formed.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forfragmenting nucleic acids.

In some embodiments, methods for fragmenting nucleic acids comprisecontacting a plurality of transpososome complexes with a plurality oftarget polynucleotides.

In some embodiments, the plurality of transpososome complexes iscontacted with the plurality of target polynucleotides in an in vitroreaction.

In some embodiments, the plurality of transpososome complexes iscontacted with the plurality of target polynucleotides in a singlereaction mixture. For example, the single reaction mixture can becontained in a single reaction vessel or a single well.

In some embodiments, in vitro methods for fragmenting nucleic acidscomprise contacting in a single reaction mixture a plurality oftranspososome complexes with a plurality of target polynucleotides.

In some embodiments, at least one transpososome complex in the pluralityof transpososome complexes comprises a first transposon end sequence anda second transposon end sequence.

In some embodiments, at least one transpososome complex in the pluralityof transpososome complexes comprises a first transposon end sequencehaving at least one modification. Optionally, the at least onemodification includes a lesion such as a nick, gap, apurinic site orapyrimidinic site.

In some embodiments, at least one transpososome complex in the pluralityof transpososome complexes comprises a second transposon end sequencehaving at least one modification. Optionally, the at least onemodification includes a lesion such as a nick, gap, apurinic site orapyrimidinic site.

In some embodiments, at least one transpososome complex in the pluralityof transpososome complexes comprises a first or a second transposon endsequence that lacks a modification (e.g., a lesion such as a nick, gap,apurinic site or apyrimidinic site).

In some embodiments, the first and the second transposon end sequenceshave identical or different sequences.

In some embodiments, in vitro methods for fragmenting nucleic acidscomprise transposing the first or the second transposon end sequencesinto the target DNA molecule and fragmenting the target DNA molecule,and joining a first end of a DNA fragment to the first transposon endsequence or the second transposon end sequence.

In some embodiments, in vitro methods for fragmenting nucleic acidscomprise transposing the first and the second transposon end sequencesinto the target DNA molecule and fragmenting the target DNA molecule,and joining a first end of a DNA fragment to the first transposon endsequence and optionally joining a second end of the DNA fragment to thesecond transposon end sequence.

In some embodiments, at least one end of a fragmented DNA molecule isjoined to the first transposon end sequence having at least onemodification. Optionally, the at least one modification includes alesion such as a nick, gap, apurinic site or apyrimidinic site.

In some embodiments, at least one end of a fragmented DNA molecule isjoined to the second transposon end sequence having at least onemodification. Optionally, the at least one modification includes alesion such as a nick, gap, apurinic site or apyrimidinic site.

In some embodiments, the fragmented DNA molecule is joined at a firstend to a first transposon end sequence, and is joined at a second end toa second transposon end sequence, and the first and the secondtransposon end sequences have identical or different sequences.

In some embodiments, one or more stabilizing agents is added before,during or after the transpososome complexes are formed.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forfragmenting DNA in vitro, comprise: (a) forming a plurality oftranspososome complexes by contacting in a single reaction mixture (i) aplurality of transposases, (ii) a plurality of polynucleotidescontaining a first transposon end sequence, wherein the first transposonend sequence is capable of binding to a transposase from the pluralityof transposases, and (iii) a plurality of polynucleotides containing asecond transposon end sequence, wherein the second transposon endsequence is capable of binding to a transposase from the plurality oftransposases, wherein the first transposon end sequence contains atleast one modification, including a lesion such as at least one nick,gap, apurinic site or apyrimidinic site, and wherein the secondtransposon end sequence optionally contains at least one modification,including a lesion such as at least one nick, gap, apurinic site orapyrimidinic site, (b) contacting the plurality of transpososomecomplexes with a plurality of target nucleic acids (e.g., target DNA),and (c) transposing the first and the second transposon end sequencesinto the target DNA molecule and fragmenting the target DNA molecule,and joining a first end of a DNA fragment to the first transposon endsequence and optionally joining a second end of the DNA fragment to thesecond transposon end sequence. Optionally, the transposome complexesmay contain a plurality of transposases, including two, three, four ormore transposases. Optionally, one or more stabilizing agents is addedafter the transpososome complexes are formed (e.g., before step (b)).Optionally, the first and the second transposon end sequences haveidentical or different sequences.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, which further comprise: producing at least one fragmented DNAmolecule, by transposing the first and the second transposon endsequences into the target DNA molecule and fragmenting the target DNAmolecule, and joining a first end of a DNA fragment to the firsttransposon end sequence and optionally joining a second end of the DNAfragment to the second transposon end sequence. Optionally, the firstand the second transposon end sequences, which are joined to the ends ofthe target DNA molecule, have identical or different sequences.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, which further comprise: forming at least onetranspososome/target DNA complex by contacting the plurality oftranspososome complexes with a plurality of target nucleic acidmolecules. Optionally, the plurality of target nucleic acid moleculescomprises DNA molecules.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, which further comprise: producing at least one fragmented DNAmolecule, by transposing the first and the second transposon endsequences into the target DNA molecule and fragmenting the target DNAmolecule, and joining a first end of a DNA fragment to the firsttransposon end sequence and optionally joining a second end of the DNAfragment to the second transposon end sequence.

Optionally, the at least one fragmented DNA molecule includes a firsttransposon end sequence having at least one modification, including alesion such as at least one nick, gap, apurinic site or apyrimidinicsite.

Optionally, the at least one fragmented DNA molecule includes a secondtransposon end sequence having at least one modification, including alesion such as at least one nick, gap, apurinic site or apyrimidinicsite.

Optionally, the first and the second transposon end sequences haveidentical or different sequences.

In some embodiments, an improved workflow can include reducing oreliminating separate steps to remove the transposase enzymes that mayinhibit a subsequent step. It is postulated that the transposase enzymeremains bound to the DNA fragments (which are joined at the ends to thetransposon end sequences), and inhibits a subsequent PCR reaction. Oneadvantage of conducting a transposon-mediated DNA fragmentation reactionwith the transposon end sequence described herein, is that the firstand/or second transposon end sequence having at least one nick or gap,which may reduce the number of hydrogen bonds between the first andsecond strands of the transposon end sequences, which may lead todissociation of the single-stranded terminal portion of the transposonend sequence without the need for a separate removal or extraction step.The absence of the terminal portion of the transposon end sequence maylead to dissociation of the transposase enzyme from the transposon endsequence after the transposon-mediated fragmentation step. A PCRreaction can be conducted in the same reaction mixture (and in the samereaction vessel) once the terminal portion of the transposon endsequence and the transposase dissociate from the fragmented DNA. Since aseparate chemical extraction step is not necessary, and the fragmentedDNA need not be transferred to a separate reaction vessel, the workflowcan be streamlined and automated.

In some embodiments, methods for preparing a plurality of transpososomecomplexes, or methods for fragmenting DNA in vitro, further comprise:reducing the length of the first transposon end sequence which is joinedto the target DNA, by truncating a terminal portion of the attackingstrand at the location of the first nick (i.e., disintegration).

In some embodiments, methods for preparing a plurality of transpososomecomplexes, or methods for fragmenting DNA in vitro, further comprise:reducing the length of the first transposon end sequence which is joinedto the target DNA, by truncating a terminal portion of the non-attackingstrand at the location of the second nick (i.e., disintegration).

In some embodiments, methods for preparing a plurality of transpososomecomplexes, or methods for fragmenting DNA in vitro, further comprise:reducing the length of the second transposon end sequence which isjoined to the target DNA, by truncating a terminal portion of theattacking strand at the location of the first nick (i.e.,disintegration).

In some embodiments, methods for preparing a plurality of transpososomecomplexes, or methods for fragmenting DNA in vitro, further comprise:reducing the length of the second transposon end sequence which isjoined to the target DNA, by truncating a terminal portion of thenon-attacking strand at the location of the second nick (i.e.,disintegration).

In some embodiments, any one or any combination of the following stepscan be conducted manually or by automation, including: preparing aplurality of transpososome complexes, forming a plurality oftranspososome/target DNA complexes, producing at least one fragmentedDNA molecule, amplifying the fragmented DNA molecule (e.g., via PCR),denaturing the amplified target DNA, immobilizing the plurality ofsingle-stranded fragmented DNA to a support, sequencing, and/orfragmenting the target DNA. For example, any reagents employed toconduct any of these steps can be reacted together (contacted) in one ormore reaction vessels. Non-limiting examples of the reagents include:transposases, first transposon end sequences, second transposon endsequences, target DNA molecules, PCR reagents (e.g., amplificationpolymerase, primers and/or nucleotides), sequencing reagents (e.g.,sequencing polymerase, primers and/or nucleotides), activating agents(e.g., magnesium and/or manganese) and/or stabilizing agents.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, further comprise: controlling the average length of thefragmented DNA molecules which can be achieved by any one or anycombination of: (i) varying the amount of transpososome complexes whichis contacted with the plurality of target DNA, (ii) varying the amountof target DNA which is contacted with the transpososome complexes, (iii)varying the amount of time of the transposition reaction, and/or (iv)varying the location of the nick or gap on the transposon end sequence.

In some embodiments, methods for preparing a plurality of transpososomecomplexes, or methods for fragmenting DNA in vitro, further comprise:amplifying the at least one fragmented DNA molecule to produce amplifiedfragmented DNA molecules.

In some embodiments, the amplifying step can be conducted by apolymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202both granted to Mullis); ligase chain reaction (LCR) (Barany 1991Proceedings National Academy of Science USA 88:189-193, Barnes 1994Proceedings National Academy of Science USA91:2216-2220); or isothermalself-sustained sequence reaction (Kwoh 1989 Proceedings National Academyof Science USA 86:1173-1177, WO 1988/10315, and U.S. Pat. Nos.5,409,818, 5,399,491, and 5,194,370); or recombinase polymeraseamplification (RPA) (U.S. Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos.5,273,881 and 5,670,316 both to Sena, and U.S. Pat. Nos. 7,270,981,7,399,590, 7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000,8,062,850, and 8,071,308).

In some embodiments, amplifying the at least one fragmented DNA moleculecomprises: contacting the at least one fragmented DNA molecule with (i)one or more primers that hybridize with at least a portion of thefragmented DNA molecule or a sequence that is complementary to thefragmented DNA molecule, (ii) one or more polymerases, and (iii) one ormore nucleotides. Optionally, the amplifying can be conducted underisothermal or thermo-cycling conditions. Optionally, the one or morepolymerases comprise thermo-stable or thermal-labile polymerases.Optionally, the amplifying can be conducted in the presence of bovineserum albumin (BSA).

In some embodiments, the amplifying step is conducted in the samereaction mixture, and in the same reaction vessel, that is used toconduct the nucleic acid fragmentation step. The reaction mixture usedfor fragmenting DNA molecules, using any of the transpososome complexesor any of the transpososome/target nucleic acid complexes describedherein, can also be used to amplify the fragmented DNA molecule. Forexample, transpososome complexes may be contacted with target DNAmolecules in a reaction mixture containing a buffer (e.g., Tris-HCl), asalt (e.g., NaCl and/or KCl), a detergent (e.g., TritonX-100), and anactivating cation (e.g., magnesium or manganese) to fragment the targetDNA molecules. Optionally, the activating agent can be added separatelyto start the amplification reaction. Optionally, components foramplifying nucleic acids (e.g., primers, polymerase and nucleotides) areadded to the fragmentation reaction mixture, using the same reactionvessel, to amplify the fragmented DNA molecules.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, further comprise: denaturing the fragmented DNA molecules, orthe amplified target DNA, to produce a plurality of single-strandedfragmented DNA. Optionally, the fragmented DNA molecules can bedenatured using chemicals or heat.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, further comprise: attaching a plurality of fragmented DNA(single-stranded or double-stranded) to a support. In some embodiments,the plurality of fragmented DNA attached to a support includesattachment to the surface of a support, or to the interior scaffold ofthe support. In some embodiments, the plurality of fragmented DNA ishybridization to capture oligonucleotides that are attached to thesupport. In some embodiments, the plurality of fragmented DNA isattached to the support by a chemical compound without hybridization tocapture oligonucleotides. In some embodiments, any chemical compoundthat can be used to attach nucleic acids to a support, can be used toattach fragmented DNA molecules or capture oligonucleotides to thesupport. For example, the support can be coated with an acrylamide,carboxylic or amine compound for attaching nucleic acids (e.g., captureoligonucleotides or fragmented DNA). In another example, amino-modifiednucleic acids can be attached to a support that is coated with acarboxylic acid. In some embodiments, amino-modified nucleic acids canbe reacted with ethyl (dimethylaminopropyl) carbodiimide (EDC) or EDACfor attachment to a carboxylic acid coated support (with or withoutN-hydroxysuccinimide (NHS)). In yet another example, nucleic acids canbe immobilized to an acrylamide compound coating on a support. In someembodiments, the support can be coated with an avidin-like compound(e.g., streptavidin) for binding biotinylated nucleic acids.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, further comprise: sequencing the plurality of single-strandedfragmented DNA with a massively parallel sequencing reaction.

In some embodiments, the massively parallel sequencing reactioncomprises incorporating a nucleotide (e.g., a nucleic acidsequence-by-synthesis reaction) or ligation-based reaction (e.g., SOLiDsequencing, Applied Biosystems/Life Technologies).

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the massively parallel sequencing reaction comprisesproviding a surface having a plurality of reaction sites (e.g., array).Optionally, the plurality of reaction sites is arranged in an organizedor random pattern.

In some embodiments, at least one reaction site is operatively linked toone or more sensors.

In some embodiments, the one or more sensors detect at least onebyproduct or cleavage product of a nucleotide incorporation reaction.

In some embodiments, the byproduct or cleavage product of a nucleotideincorporation reaction includes ions (e.g., hydrogen ions), protons,phosphate groups, including pyrophosphate groups.

In some embodiments, the one or more sensors detect a change in ions,hydrogen ions, protons, phosphate groups, including pyrophosphategroups.

In some embodiments, the one or more sensors comprise a field effecttransistor (FET).

Optionally, the sensor comprises an ion-sensitive field effecttransistor (ISFET).

In some embodiments, a plurality of fragmented DNA molecules can beproduced by conducting any method and any transposome complex describedherein.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the plurality of first transposon end sequencesincludes a plurality of double-stranded polynucleotides having a firstand second strand.

In some embodiments, the first transposon end sequences include firststrands containing at least one modification (e.g., at least onemodification includes a lesion such as a nick, gap, apurinic site orapyrimidinic site).

In some embodiments, the first transposon end sequences include secondstrands which optionally contain at least one modification (e.g., atleast one modification includes a lesion such as a nick, gap, apurinicsite or apyrimidinic site).

In some embodiments, the first transposon end sequences includedouble-stranded polynucleotides containing first strands having at leastone modification, and second strands containing at least onemodification, where the modification(s) on the first strands are locatedat a different position than the modification(s) on the second strands.Optionally, the modifications include a lesion such as a nick, gap,apurinic site or apyrimidinic site.

In some embodiments, the first transposon end sequences includedouble-stranded polynucleotides containing first and second strands,where at least one first strand or at least one second strand lacks amodification.

In some embodiments, the first transposon end sequence includes anidentification sequence (e.g., barcode sequence), a primer extensionsequence, or a sequence which is complementary to a primer extensionsequence. Optionally, a primer extension sequence includes anamplification primer sequence or a sequencing primer sequence.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the plurality of second transposon end sequencesincludes a plurality of double-stranded polynucleotides having a firstand second strand.

In some embodiments, the second transposon end sequences include firststrands containing at least one modification (e.g., at least onemodification includes a lesion such as a nick, gap, apurinic site orapyrimidinic site).

In some embodiments, the second transposon end sequences include secondstrands which optionally contain at least one modification (e.g., atleast one modification includes a lesion such as a nick, gap, apurinicsite or apyrimidinic site).

In some embodiments, the second transposon end sequences includedouble-stranded polynucleotides containing first strands having at leastone modification, and second strands containing at least onemodification, where the modification(s) on the first strands are locatedat a different position than the modification(s) on the second strands.Optionally, the modifications include a lesion such as a nick, gap,apurinic site or apyrimidinic site.

In some embodiments, the second transposon end sequences includedouble-stranded polynucleotides containing first and second strands,where at least one first strand or at least one second strand lacks amodification.

In some embodiments, the second transposon end sequence includes anidentification sequence (e.g., barcode sequence), a primer extensionsequence, or a sequence which is complementary to a primer extensionsequence. Optionally, a primer extension sequence includes anamplification primer sequence or a sequencing primer sequence.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the first and the second transposon end sequences haveidentical or different sequences.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the first transposon end sequence comprises adouble-stranded polynucleotide having a first attacking strand and afirst non-attacking strand.

In some embodiments, the first attacking strand includes a least onemodification, including a lesion such as at least one nick, gap,apurinic site or apyrimidinic site.

In some embodiments, the first non-attacking strand includes a least onemodification, including a lesion such as at least one nick, gap,apurinic site or apyrimidinic site.

Optionally, the first transposon end sequence includes a first attackingstrand and a first non-attacking strand, wherein the first attackingstrand and/or the first non-attacking strand include a least onemodification, including a lesion such as at least one nick, gap,apurinic site or apyrimidinic site.

In some embodiments, the first transposon end sequences include a firstattacking strand and a first non-attacking strand, wherein the firstattacking strand or the first non-attacking strand lacks a modification.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the second transposon end sequence comprises adouble-stranded polynucleotide having a second attacking strand and asecond non-attacking strand.

In some embodiments, the second attacking strand includes a least onemodification, including a lesion such as at least one nick, gap,apurinic site or apyrimidinic site.

In some embodiments, the second non-attacking strand includes a leastone modification, including a lesion such as at least one nick, gap,apurinic site or apyrimidinic site.

Optionally, the second transposon end sequence includes a secondattacking strand and a second non-attacking strand, wherein the secondattacking strand and/or the second non-attacking strand include a leastone modification, including a lesion such as at least one nick, gap,apurinic site or apyrimidinic site.

In some embodiments, the second transposon end sequences include a firstattacking strand and a first non-attacking strand, wherein the firstattacking strand or the first non-attacking strand lacks a modification.

Optionally, the first and/or the second transposon end sequence includesan attacking strand having a nick which is located at any position,including after the sixth, eighth, tenth, fourteenth, sixteenth,eighteenth, nineteenth or twenty-seventh nucleotide from the 3′ end ofthe attacking strand (see FIG. 8).

Optionally, the first and/or the second transposon end sequence includesa non-attacking strand having a nick which is located at any position,including after the eleventh, thirteenth, twenty-fifth or twenty-seventhnucleotide from the 5′ end of the attacking strand (see FIG. 8).

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the transpososome complexes include a transposaseenzyme comprising any transposase enzyme, including a DDE transposaseenzyme such as a prokaryotic transposase enzyme from ISs, Tn3, Tn5, Tn7,and Tn10, bacteriophage transposase enzyme from phage Mu (Nagy andChandler 2004, reviewed by Craig et al. 2002; U.S. Pat. No. 6,593,113),and eukaryotic “cut and paste” transposase enzymes (Jurka et al. 2005;Yuan and Wessler 2011). In some embodiments, the transposase enzymesinclude retroviral transposases, such as HIV (Dyda et al. 1994; Haren etal. 1999; Rice et al. 1996; Rice and Baker 2001).

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein the transpososome complexes include a first and/or thesecond transposon end sequence comprising any transposon sequence (e.g.,a transposon end sequence) from a prokaryotic insertion sequencesincluding (ISs), Tn3, Tn5, Tn7, and Tn10, bacteriophage included phageMu (Nagy and Chandler 2004, reviewed by Craig et al. 2002), andeukaryotic “cut and paste” transposons (Jurka et al. 2005; Yuan andWessler 2011). In some embodiments, the first and/or the secondtransposon end sequence includes any transposon sequence fromretroviruses such as HIV (Dyda et al. 1994; Haren et al. 1999; Rice etal. 1996; Rice and Baker 2001).

In some embodiments, the first transposon end sequence comprises a MuAtransposon end sequence, Mos1 transposon end sequence, Vibrio harveytransposon end sequence, or Tn5 transposon end sequence.

In some embodiments, the second transposon end sequence comprises a MuAtransposon end sequence, Mos1 transposon end sequence, Vibrio harveytransposon end sequence, or Tn5 transposon end sequence.

In some embodiments, the transposase enzyme comprises a MuA, Mos1,Vibrio harvey, ISs, Tn3, Tn5, Tn7, or Tn10 transposase.

In some embodiments, the first and second transposon end sequencescomprise MuA transposon end sequences and the transposase comprises aMuA transposase.

In some embodiments, the first and second transposon end sequencescomprise Mos1 transposon end sequences and the transposase comprises aMos1 transposase.

In some embodiments, the first and second transposon end sequencescomprise Vibrio harvey transposon end sequences and the transposasecomprises a Vibrio harvey transposase.

In some embodiments, the first and second transposon end sequencescomprise Tn5 transposon end sequences and the transposase comprises aTn5 transposase.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, forpreparing a plurality of transpososome complexes, or for fragmenting DNAin vitro, wherein any combination of reagents used to conduct any stepor reaction described herein can be deposited into one or more reactionvessels in any order, including sequentially or substantiallysimultaneously or a combination of both. Non-limiting examples of thereagents include: transposases, first transposon end sequences, secondtransposon end sequences, target DNA molecules, PCR reagents (e.g.,amplification polymerase, primers and/or nucleotides), sequencingreagents (e.g., sequencing polymerase, primers and/or nucleotides),activating agents and/or stabilizing agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of fragmented target DNA with uniformtransposon ends when a standard full length (native) transposon end,transposon end with pre-nicked attacking strand, and transposon end withboth pre-nicked strands are used.

FIG. 2 shows Agilent 2100 Bioanalyzer curves of fragmented Escherichiacoli genomic DNA (gDNA) when a full length (native) transposon end andvarious pre-nicked transposon ends were used for MuA transpososomeformation and subsequent DNA fragmentation.

FIG. 3 shows Agilent 2100 Bioanalyzer curves of Human gDNA, HeLa dscDNA, S. aureus gDNA, E. coli gDNA and T. thermophilus gDNA fragmentedusing MuA-pre-nicked transposon end complex.

FIG. 4 shows Agilent 2100 Bioanalyzer curves of fragmented E. coli gDNAwhich displays DNA fragment length dependence on the amount ofMuA-pre-nicked transposon end complex (0.25 μl-2 μl) used in the DNAfragmentation reaction.

FIG. 5 shows Agilent 2100 Bioanalyzer curves of E. coli gDNA fragmentedusing 44-6 pre-nicked transposon end containing MuA transpososome (curveA) and final size-selected DNA library with ligated adaptors (curve B),used for DNA template preparation and subsequent sequencing on IonTorrent PGM.

FIG. 6 shows Ion Torrent PGM DNA library, prepared from E. coli gDNAfragmented with 44-6 pre-nicked transposon end containing MuAtranspososome, run summary fragments: ISP density MAP, chip welldetails, read length details, alignment summary, raw accuracy.

FIG. 7 shows Agilent 2100 Bioanalyzer curves of fragmented (using nativetransposon ends and 34-16 pre-nicked transposon ends) and then amplifiedhuman gDNA.

FIG. 8 shows the structure of transposon ends designed for DNAfragmentation experiments using MuA transpososome containing nickedtransposon ends and gap-containing transposon ends (examples 4-6).

FIG. 9 shows Agilent 2100 Bioanalyzer curves of E. coli gDNA fragmentedusing MuA transpososomes containing native transposon ends, 38-12 nickedtransposon ends and gaped transposon ends (Gap42-6, Gap40-8).

FIGS. 10 (A) and (B) show Agilent 2100 Bioanalyzer curves of fragmentedE. coli gDNA illustrating DNA fragment length dependence on thetransposon end structure assembled into MuA transpososome, amount (0.5μl, 1.5 μl) of MuA transpososome containing native transposon ends,38-12 nicked transposon ends or 42-6 transposon ends with gap and invitro transposition reaction time (1.5 min, 5 min, 10 min).

FIG. 11 shows Agilent 2100 Bioanalyzer curves of fragmented E. coli gDNAillustrating the efficiency of in vitro transposition reaction usingfixed amount (1.5 μl) of MuA transpososome containing native transposonend or 42-6 gaped transposon end, prolonged in vitro transpositionreaction time (30 min), and lower target DNA input (25 ng, 50 ng, and100 ng).

FIG. 12 shows Agilent 2100 Bioanalyzer curves of fragmented E. coli gDNAillustrating the variable of in vitro transposition reaction time forMuA-native transposon end complex and MuA-42-6 gaped transposon endcomplex while the amount of input DNA (100 ng) and MuA transpososome (3μl) containing either native transposon end or 42-6 gaped transposon endis the same.

FIG. 13 represents deviations from the theoretical expected distributionof nucleotide replacements (degeneration) in MuA transposon endsequence.

FIG. 14A-E show Agilent 2100 Bioanalyzer profiles of fragmented E. coligDNA obtained with transposome complexes comprising modified(degenerate) transposon ends.

FIG. 15 shows EMSA analysis of the MuA-modified transposon complexes.

FIG. 16 shows Agilent 2100 Bioanalyzer curves of fragmented (usingnative transposon ends and 34-16 pre-nicked transposon ends) and thenamplified human gDNA.

DESCRIPTION

The invention relates to the field of controlled fragmentation ofnucleic acids.

Artificial transposons containing nicked DNA strands and their use inapplications including, but not limited to, Next Generation Sequencing(NGS) library preparation.

A method for generation of nucleic acid fragments of desired lengthbased on in vitro transposition reaction in the presence of atransposase, and a specially designed pair of transposon ends, eitherone or both harboring a modified sequence. A modified sequenceencompasses both degenerate sequences, defined as a sequence other thanwild type, and a sequence containing an artificially introduced apurinicsite, apyrimidinic site, nick, or nucleotide gap, which may generally betermed an artificially introduced lesion. A transposase and a transposonend containing a modified sequence and/or modified to containapurinic/apyrimidinic sites, nicks, or gaps are assembled intocatalytically active complexes and are used to enzymatically shear anucleic acid sample in a controlled fragmentation process that yieldsdesired average nucleic acid fragment size. Such complexes of atransposase and a modified transposon end, when used in generation ofDNA sequencing templates, offer advantages compared to currentlyavailable transposon-based DNA fragmentation kits. Such advantagesinclude shortened stretches of transposon end sequences, ability to addspecific adapters, and ability to be used in applications whereproduction of asymmetrically tailed DNA fragments, i.e., possessing thetransposed transposon end of the full length only at one end ofprocessed DNA fragment, is preferred. The invention is further directedto transposon nucleic acids comprising a transposon DNA end sequenceharboring a modified sequence.

Transposons containing modified sequences, either in an attacking strandor in both DNA strands, can be used for transposon/transposase complexformation and subsequent fragmentation of DNA of interest. As a result,transposition reaction products (sheared DNA fragments) contain uniformterminal sequences whose length and structure is anticipated by amodified transposon end sequence position in primary transposons, andwhich is considerably shorter compared to using full-length artificialtransposons. Introduction of a modified transposon end sequence atspecific positions of transposons ensures considerably higher PCRefficiency and allows formation of sticky ends of known structure thatmay be used for ligation with adapters carrying complementary stickyends.

The inventive transposase-based DNA shearing approach is a powerfulalternative to other DNA shearing techniques. It requires very lowamounts of input DNA material. In addition to DNA fragmentation, itresults in specific uniform DNA sequences at both ends of the resultingDNA fragments.

In embodiments, sequences originating from transposons are used forannealing PCR primers. For a transposase-based method, uniform fragmentends are generally long enough for primer annealing, e.g., about 10nt-25 nt, if PCR is intended to be used for amplification of reactionproducts. Longer complementary ends of resulting fragments lead toformation of very stable secondary DNA structures, known as“stem-loops”, which prevent primer annealing and drastically inhibit PCRefficiency. When full length transposons are used, terminalcomplimentary ends are 50 bp long and thus require special conditionsfor subsequent gap-filling and strand displacement steps to ensuresufficiently high PCR efficiency.

Such drawbacks are solved using transposons with internal modifiedsequences in attacking DNA strands, leading to the formation ofcomplementary ends of optimal length. Depending on experimentalconditions, these complimentary ends may vary from a few to several tensof nucleotides, based upon the introduction of modified sequences intoappropriate positions of transposons used for DNA shearing. For example,in PCR assisted adaptor addition reactions, longer fragments arepreferred over shorter fragments when DNA was sheared with transposonscontaining modified sequences or lesions, which provided an advantagewhen recently developed 400 bp or longer sequencing protocols arefurther applied. However, if ligation is intended to be used for addingspecific adapters, transposons for DNA fragmentation should havemodified sequences either in the attacking or in both transposonstrands. The latter modification of transposons results in theappearance of sticky ends at both ends of DNA fragments which aresuitable for ligation with adapters having complementary sticky ends.Products of either PCR or ligation reaction or both may be used invarious assays, including but not limited to NGS and microarrayanalysis.

Unless specifically defined or described differently, the invention usesthe following terms and descriptions.

The term “transposon” as used herein is a nucleic acid segment that isrecognized by a transposase or an integrase enzyme and which is anessential component of a functional nucleic acid-protein complex(“transposome complex”) capable of transposition. The inventivetransposons in one embodiment belong to class II transposable DNAelements, which use fundamentally similar reactions for their movementwithin and between genomes, namely, the transposition reaction iscatalyzed by a transposase enzyme by either a double- or single-strandedDNA intermediate and transposon DNA is translocated in the “cut andpaste” manner within genome. The term “transposon” as used herein alsoincludes all derivatives of the original transposable element, such asmini-transposons or other reiterations of minimal nucleic acid-proteincomplex capable of transposition, including but not limited to twoindividual not interconnected transposon ends, or said ends joined bysome artificial linker.

The term “transposase” as used herein refers to an enzyme that is acomponent of a functional nucleic acid-protein complex capable oftransposition and that mediates transposition.

The terms “transposon end” or “transposon end sequence” is a sequencerecognized by a transposase enzyme necessary to form a synaptic complexor a “transpososome complex”, sufficient for a subsequent transpositionevent to occur in vitro. “Sufficient for a subsequent transpositionevent to occur in vitro” means transposon end sequences necessary forboth recognition and binding of a transposase enzyme, including aterminal stretch of nucleotides of about five base pairs, the last twobase pairs being the attacking 5′-CA, these five base pairs necessaryfor the transposition reaction to occur. A transposon end andtransposase protein form a “complex” or a “synaptic complex” or a“transposome complex”, the complex capable of inserting or transposingthe transposon end into target DNA with which it is incubated in an invitro transposition reaction. Transpososomes contain multiple subunitsof a transposase protein, bound to DNA sequences from both of thetransposon's ends. These protein-DNA complexes are also called “synapticcomplexes” because they bring together the two ends of the transposonDNA. The phage Mu transposase, MuA, is monomeric in solution but forms atetramer upon binding to specific DNA recognition sites near thetransposon ends. The critical reaction steps mimicking Mu transpositioninto external target DNA can be reconstituted in vitro using MuAtransposase, 50 bp Mu R-end DNA segments, and target DNA as the onlymacromolecular components (Haapa et al. An efficient and accurateintegration of mini-Mu transposons in vitro: A general methodology forfunctional genetic analysis and molecular biology applications. NucleicAcids Res 27 (1999) 2777-2784).

The term “adaptor” as used herein refers to a non-target nucleic acidcomponent, generally DNA, that provides a means of addressing a nucleicacid fragment to which it is joined. For example, an adaptor comprises anucleotide sequence that permits identification, recognition, and/ormolecular or biochemical manipulation of the DNA to which the adaptor isattached (e.g., by providing a site for annealing an oligonucleotide,such as a primer for extension by a DNA polymerase, or anoligonucleotide for capture or for a ligation reaction).

The term “equimolar concentration” as used herein refers to transposaseprotein-transposon nucleic acid ratio enabling formation of completelyassembled transposon complexes where all complex partners are utilizedand no excessive complex partners remain free in solution. For the Mutransposition system such ratio represents four MuA transposase proteinmolecules and two Mu transposon end sequences that are able to interactwith MuA, while for the Tn5 system such ratio is two Tn5 transposaseprotein molecules and two Tn5 transposon end sequences. In the inventivemethod, transposome assembly is performed in more concentratedtransposase/transposon end reaction mixture (in concentrations thatcorrespond to 4 transposase molecules per 2 transposon ends (“equimolarconcentrations”)) optimized for transposome complex formation. Afterassembly, the preformed complexes are diluted and target DNA is added. Areaction mixture with target DNA is suboptimal for complex formation, soin addition to the lack of transposase turnover, this is another factorthat leads to exhaustion of preformed active transposome complexes andthe reaction stops at certain level of DNA degradation.

In some embodiments, a “support” comprises a planar surface, as well asconcave, convex, or any combination of surfaces thereof. In someembodiments, a “support” includes a bead, particle, microparticle,sphere, filter, flowcell, well, microwell, groove, channel reservoir,gel or inner wall of a capillary. Optionally, the support includes theinner walls of a capillary, a channel, a well, microwell, groove,channel, reservoir. Optionally, the support includes include texture(e.g., etched, cavitated, pores, three-dimensional scaffolds or bumps).Optionally, the support can be porous, semi-porous or non-porous.Optionally, the support includes one or more beads having cavitation orpores, or can include three-dimensional scaffolds. Optionally, thesupport includes an Ion Sphere™ particle (from Ion Torrent, part of LifeTechnologies, Carlsbad, Calif.). Optionally, the particles have anyshape including spherical, hemispherical, cylindrical, barrel-shaped,toroidal, rod-like, disc-like, conical, triangular, cubical, polygonal,tubular, wire-like or irregular. In some embodiments, the support can bemade from any material, including glass, borosilicate glass, silica,quartz, fused quartz, mica, polyacrylamide, plastic polystyrene,polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA),polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics,silicon, semiconductor, high refractive index dielectrics, crystals,gels, polymers, or films (e.g., films of gold, silver, aluminum, ordiamond). In some embodiments, the support can be magnetic orparamagnetic. In some embodiments, the support includes paramagneticbeads attached with streptavidin (e.g., Dynabeads™ M-270 fromInvitrogen, Carlsbad, Calif.). Optionally, the bead or particle can havean iron core, or comprise a hydrogel or agarose (e.g., Sepharose™).Optionally, the support is coupled to at least one sensor that detectsphysicochemical byproducts of a nucleotide incorporation reaction, wherethe byproducts include pyrophosphate, hydrogen ion, charge transfer, orheat.

Interaction of various transposases with their substrate DNA (transposonends) are described in the prior art to the extent that allows oneskilled in the art to determine borders of transposon ends for varioustransposome complexes (Montano and Rice 2011. Moving DNA around: DNAtransposition and retroviral integration. Curr. Opin. Struct. Biol. 21,370-378). The scientific literature may reference transposon ends as aprimary substrate for a transposase protein, which is necessary fortransposome complex assembly. However, only a fully assembledtransposome can attack target DNA, so the target DNA is a substrate forassembled transposome complex, but not for transposase enzyme.

Conventional DNA sequencing methods are currently being replaced by socalled “next-generation” technologies or “massive parallel sequencing”platforms that allow millions of nucleic acid molecules to be sequencedsimultaneously. These methods rely on a sequencing-by-synthesisapproach, while other platforms are based on a sequencing-by-ligationtechnology. All of these new technologies rely on a pool of sequencingtemplates (DNA library), which later may be multiplied by use of DNAamplification techniques.

There are two main methodologies in DNA library preparation. So-calledconventional DNA library preparation procedures include DNAfragmentation (hydroshearing, sonication, nebulization, or enzymaticshearing) followed by DNA repair and end-polishing (blunt-end orA-overhang), and finally platform-specific adaptor ligation.Transposon-based DNA library preparation procedures use in vitrotransposition to prepare sequencing-ready DNA libraries: during in vitrotransposition catalyzed by transposase-transposon end complex, strandtransfer occurs via random, staggered double-strand DNA breaks in thetarget DNA and covalent attachment of the 3′ end of the transferredtransposon strand to the 5′ end of the target DNA. When two transposonends which participate in the in vitro transposition reaction are notinterconnected, the target DNA is fragmented and the transferred strandof the transposon end oligonucleotide is covalently attached to the 5′end of the DNA fragment. Independent tags can also be added to thefragmented DNA by appending the transposon end sequence with anengineered adaptor sequence. After extension, the sequencing adaptorsenable amplification by emulsion PCR (emPCR), bridge PCR (bPCR), andother methods.

Commercial conventional and transposon based DNA library preparationmethods in the form of DNA library preparation kits are available (e.g.,Illumina, Life Technologies, New England Biolabs), but these facelimitations: multi-step protocols require numerous DNA manipulationsteps that result in long and laborious workflow and may result insignificant DNA sample loss and limited throughput.

Transposon based DNA library preparation methods, although in generaldemand less hands-on time and are more convenient for the user, alsohave limitations: in vitro transposition products having complementarytransposon end sequences at both ends tend to form intramolecular loopstructures when denatured to single stranded DNA. This is particularly aproblem when the fragmented DNA is subjected to PCR amplification.

In addition, massive parallel sequencing platforms typically requirethat the initially long polynucleotide chains be reduced to smallernucleic acid fragments having average length in base pairs as specifiedby the operational setting of the sequence reader. Current sequencingread lengths vary from 50 bp to 1000 bp. For some massive parallelsequencing platforms mate-pair sequencing library preparation methodsdemanding nucleic acid fragments from 5 kb to 40 kb are applied.Conventional nucleic acid fragmentation methods, such as enzymaticdigestion, nebulization, hydroshear, and sonication are based on randomnucleic acid shearing, thus required nucleic acid fragments havinglength corresponding to operational setting of the sequence reader needto be additionally purified from the mixture of nucleic acid fragmentsby various size selection protocols.

There still is a need for methods that enable fragmentation of a DNAsample to a desired predefined average DNA fragment size, and thatfacilitate downstream handling of the fragmented DNA obtained from thein vitro transposition step.

Methods were suggested to overcome self-annealing of complementarytransposon end sequences resulting from in vitro transposition event.For example, in Nextera DNA Sample Preparation Kits that employ Tn5transposase-transposon end complexes, transposon ends are appended withsequencing primer sequences that are not complementary.

Grunenwald U.S. Published Patent Application No. 2010/0120098 disclosemethods for using a transposase and a transposon end for generatingextensive fragmentation and 5′-tagging of double-stranded target DNA invitro. The method is based on use of a DNA polymerase for generating 5′-and 3′-tagged single-stranded DNA fragments after fragmentation withoutperforming PCR amplification reaction. Tagged transposon ends aredisclosed, but the actual transposon end sequence of the usedtransposons corresponds to the native Tn5 transposon sequence. The tagdomain combined with the native transposon end can comprise a sequenceor structure of a cleavable site, with the method comprising a step ofincubating the tagged DNA fragments obtained from the fragmentation stepwith a cleavage enzyme. The application describes transposon ends havingthe cleavage site in the tag sequence that is attached to the 5′-end ofthe transposon end sequence, but not in the transposon end sequenceitself.

Kavanagh U.S. Published Patent Application No. 20130017978 teach methodsto truncate transposon ends after DNA fragmentation thereby making themnon complementary. The methods comprise the steps of (a) initiating anin vitro transposition reaction in the presence of a transposon end,transposase enzyme, and target DNA, wherein the transposon end comprisesa transposon end sequence recognizable by a transposase, the transposonend sequence comprising a modified position or modified positions, wherethe modified position or positions introduce(s) a cleavage site into thetransposon end sequence, and where the in vitro transposition reactionresults in fragmentation of the target DNA and incorporation of thetransposon end into the 5′ ends of the fragmented target DNA; and (b)incubating the fragmented target DNA with an enzyme specific to thecleavage site so the transposon ends incorporated into the fragmentedtarget DNA are cleaved at the cleavage site. Examples of suchmodification/cleavage are provided: transposons containing uracil can betruncated using UDG and subsequent heat or endonuclease treatment;transposons containing m5C can be truncated using methylation sensitiverestriction endonuclease SgeI; and transposons containing RNA/DNA hybridparts can be truncated using RNAse H.

Although these approaches may be used for efficient transposon endtruncation, they have limitations. Use of uracil, m5C or RNA can impedetransposase-transposon end complex formation or inhibit its activity invitro (DNA fragmentation capability) due to unnatural transposon endstructure. The need to introduce such modifications into transposon endsincreases the cost of synthesis of transposon end oligonucleotides.Transposon end truncation is an enzymatic process that requirescomplicated optimization and extends DNA library preparation workflowtime. After incubation, enzymes used for cleavage need to be inactivatedor removed to avoid possible negative impacts in later DNA librarypreparation steps.

The inventive method provides modifications introduced into certainpositions of transposon ends, the modifications including lesions suchas a nick, nucleotide gap, or a modified sequence including adegenerated transposon end sequence which, after complexing suchtransposon ends with a transposase enzyme, result in synaptic complexesthat, although with decreased affinity to the transposon end sequences,are both sufficiently stable in the in vitro transposition reactionmixture, and can perform in vitro transposition events resulting in acontrolled DNA fragmentation that reduced the DNA sample to a desiredaverage DNA fragment size. Such transposase/modified transposon end(containing apurinic/apyrimidinic site, nick, or gap) complexes may beapplied for generation of DNA fragments of predefined length, or as inthe case of DNA lesions, for generation of DNA fragments of predefinedlength that contain shortened stretches of transposon end sequences andfor production of asymmetrically tailed DNA fragments.

The possibility of MuA transposase to accommodate and process a varietyof different hairpin structured substrates containing right transposonends with MuA-binding sites R1 and R2 was addressed (Saariaho et al.,Nucleic Acids Research 34 (2006) 3139-3149). As a result, most of Muspecific hairpin substrates were generated from two oligonucleotides,and therefore these hairpin substrates contained a nick within the R1MuA binding site. A direct comparison of in vitro transpositionreactions catalyzed by MuA transposase interacting with nicked andcorresponding unnicked hairpin substrate indicated that this nick doesnot interfere with in vitro transposition reactions. The structure ofcrystallized MuA final strand transfer complex, which contained atetramer of truncated MuA proteins, two copies of the bacteriophage Muend DNA, and one target DNA was resolved (Montano et al., Nature 491(Nov. 15, 2012) 413-417). Mu end DNA duplexes were assembled to mimicthe transposition reaction product of initial bacteriophage Mutransposon DNA cleavage by MuA transposase generating pre-cleaved righttransposon ends exposing free 3′-OH groups able to attack 5′-end of thetarget DNA. Therefore, Mu end DNA duplexes were prepared by mixing inequal molar amounts four single stranded oligonucleotides containing R1and R2 binding sites for recognition and binding of four MuA proteinsresulting in a nick on each strand of the duplex at a position that doesnot interfere with transpososome assembly. MuA transposition machinerytolerates certain variability in the transposon end sequences(Goldhaber-Gordon et al. J Biol Chem. 277 (2002) 7703-12). However,neither of these publications suggested that nick- or gap-bearing, ordegenerate sequences-bearing, transposon ends assembled intocatalytically active complexes with a transposase enzyme can be appliedfor controlled DNA fragmentation, generation of DNA fragments containingshortened stretches of transposon end sequences, or production ofasymmetrically tailed DNA fragments.

Use of pre-nicked transposon ends or transposon ends with a nucleotidegap is a fast and simple alternative to enzymatic truncation. Pre-nickedtransposon ends or transposon ends with a nucleotide gap do not containany sophisticated modifications which could distort DNA structure, socomplex formation process efficiency is comparable to thatcharacteristic for a native transposon end. After an in vitrotransposition reaction, such transposon ends are held on complementaryDNA by weak hydrogen bonds and, depending on the length of thecomplementary region, either disintegrate from fragmented DNA itselfimmediately after the in vitro transposition reaction or after theinitial adapter addition PCR (AA-PCR) elongation step in those caseswhen a nick or gap was introduced far from the 3′ end of the attackingstrand. As the transposon end truncation is determined by the nick orgap position in the attacking transposon strand, it may be variedeasily. Transposon end truncation via nick or gap introduction may beused for generating shorter complementary transposon ends in the DNAlibrary construction, leading to more efficient amplification offragmented DNA, and production of asymmetrically tailed DNA fragmentspossessing the full length transposon end only at one end of processedDNA fragment.

The data provided herein demonstrate that, surprisingly, transpososomesformed from about equimolar concentrations of transposase and modifiedtransposon ends comprising a nick or a nucleotide gap or degeneratednucleotide sequences can form active transposomes. Due to lowertransposase affinity to its substrate (modified transposon end) theactual effective concentration of fully assembled transposomes is lowerthan that obtained using native transposon ends. This enables generationof DNA fragments of desired length by varying the amounts of eithertarget genomic DNA or preassembled transposome complex in thetransposition reaction mixture. It is known that DDE transposasesusually do not turn over and MuA complex is so stable that it remainstightly bound to target DNA until it is removed in an ATP-dependentfashion by ClpX protein (Gueguen et al., TRENDS in Microbiology 13(2005) 543; Nakai et al. Proc Natl Acad Sci USA. 98 (2001) 8247-54),therefore the number of transposition events in the reaction mixture isdetermined by the effective amount of preassembled transposomes assecondary transposition does not occur and the reaction is terminated(all preassembled transposome complexes added to the reaction mixtureare exhausted) and DNA fragments of a certain average length areobtained as a result. DDE transposases are ubiquitous and represent themajority of characterized transposases, whose overall catalyticmechanism is known (Mizuuchi 1992a; reviewed by Mizuuchi 1992b). Membersof the DDE transposase family carry a conserved triad of acidicresidues: a DDE motif. The three acidic residues are crucial in thecoordination of divalent metal ions required for catalysis (Kulkosky etal., 1992). The abundant DDE transposase family includes prokaryoticinsertion sequences (ISs), members of the Tn3 family of transposons, theTn7, Tn5 and Tn10 families and transposable bacteriophages such as phageMu (Nagy and Chandler 2004, reviewed by Craig et al. 2002) andeukaryotic “cut and paste” transposons (Jurka et al. 2005; Yuan andWessler 2011). The family can be extended to include retroviruses suchas HIV, which encode a catalytic integrase protein similar to the DDEtransposases (Dyda et al. 1994; Haren et al. 1999; Rice et al. 1996;Rice and Baker 2001). “Turn over rate” defines the maximum number ofsubstrate molecules that the enzyme can ‘turn over’ to product in a settime. According to the literature, DDE transposases do not turn overunder normal reaction conditions, i.e. after catalyzing one insertioninto target DNA, the transposase stays bound to target DNA and is notreleased, thus the same enzyme molecule cannot participate in theassembly of the second transposome complex and cannot catalyze thesecond transposition event. As a result, the number of transpositionevents is dependent on the effective amount of catalytically activetransposome complexes added to reaction mixture after which the reactionstops generating DNA fragments of a certain average length.

The inventive preassembled transposomes may be used as a universal andcontrolled DNA fragmentation tool enabling generation of DNA fragmentshaving predefined average length. Embodiments are illustrated by thefollowing non-limiting Examples and Figures.

One skilled in the art will find it apparent that various transposaseenzymes complexed with their relevant transposon ends containing eitheran abasic site, nick, or gap may be used to practice the full scope ofthe invention. For example, in Mos1 and Mu transpososomes, transposonends are bound by the helix-turn-helix-motifs of the bipartite DNAbinding domains. Similar architecture of the transposon DNA-bindingindicates that a gap or a nick can be efficiently accommodated in thetransposon end DNA fragment without significant loss of function ineither Mos1 transpososome, as was shown here to be the case for MuA.Another example of a suitable enzyme for practicing the invention is Tn5transposome complex. Transposon ends required to produce stable dimerictransposome complex with Tn5 transposase are disclosed in U.S. PatentApplication Publication No. 2010/0120098. It was shown that, like MuAtransposase, Tn5 transposase tolerates nucleotide substitutions, abasicsites, and even nucleotide deletions in certain positions of itstransposon end (Jilk et al. The organization of the outside end oftransposon Tn5, J. Bacteriol. 178 (1996)1671-1679) and is still able tobind to such substrates in vitro. It is therefore reasonable to expectthat this enzyme when complexed with pre-nicked transposon ends ortransposon ends containing a gap should also perform controlled DNAfragmentation disclosed in the present invention. Similarly, transposasefrom Vibrio harvey tolerates substitutions in its binding sequence IRR(EP 2,527,438 Methods and compositions for DNA fragmentation and taggingby transposases) hence, taking into account the significant homologybetween Tn5 transposase protein and Vibrio harvey transposase (40%homology), it is reasonable to expect that this transposase may also beused as one more enzyme to practice the present invention. The dataprovided herein are sufficient to teach the skilled artisan whichsequences within the transposon end have to be modified by converting todegenerate sequences to obtain transposomes with decreased affinity toits substrate-transposon ends, resulting in a slower transpositionreaction rate for the transposome complex and/or a lower transposaseaffinity to its substrate (transposon) within the transposome complex.By reducing the reaction rate and/or affinity, the transposome complexfragments the target DNA at a lower rate. The lower rate of target DNAfragmentation enhances control of the average length of the resultingDNA fragments by varying (a) an amount of transposon complex, (b) anamount of target DNA in the reaction complex, (c) incubation time of thetransposition reaction, (d) amount of the introduced degeneratesequence, (e) location of the degenerate sequence. Manipulating thereaction conditions used with the modified transposon end sequencepermits determination of reaction conditions, e.g., incubation time,that results in a particular average fragment length. Those determinedconditions can then be used to create a DNA library comprising adesired, predefined average fragment length.

In some embodiments, any fragmented nucleic acid that has been generatedaccording to the present teachings can be sequenced by any sequencingmethod, including sequencing-by-synthesis, ion-based sequencinginvolving the detection of sequencing byproducts using field effecttransistors (e.g., FETs and ISFETs), chemical degradation sequencing,ligation-based sequencing, hybridization sequencing, pyrophosphatedetection sequencing, capillary electrophoresis, gel electrophoresis,next-generation, massively parallel sequencing platforms, sequencingplatforms that detect hydrogen ions or other sequencing by-products, andsingle molecule sequencing platforms. In some embodiments, a sequencingreaction can be conducted using at least one sequencing primer that canhybridize to any portion of the nucleic acid templates, including anucleic acid adaptor or a target polynucleotide.

In some embodiments, any fragmented nucleic acid template that has beengenerated according to the present teachings can be sequenced usingmethods that detect one or more byproducts of nucleotide incorporation.The detection of polymerase extension by detecting physicochemicalbyproducts of the extension reaction, can include pyrophosphate,hydrogen ion, charge transfer, heat, and the like, as disclosed, forexample, in U.S. Pat. No. 7,948,015 to Rothberg et al, and U.S. PatentPublication No. 2009/0026082 to Rothberg et al, hereby incorporated byreference in their entireties. Other examples of methods of detectingpolymerase-based extension can be found, for example, in Pourmand et al,Proc. Natl. Acad. Sci., 103: 6466-6470 (2006); Purushothaman et al.,IEEE ISCAS, IV-169-172; Anderson et al, Sensors and Actuators B Chem.,129: 79-86 (2008); Sakata et al., Angew. Chem. 118:2283-2286 (2006);Esfandyapour et al., U.S. Patent Publication No. 2008/01666727; andSakurai et al., Anal. Chem. 64: 1996-1997 (1992), which are herebyincorporated by reference in their entireties.

Reactions involving the generation and detection of ions are widelyperformed. The use of direct ion detection methods to monitor theprogress of such reactions can simplify many current biological assays.For example, template-dependent nucleic acid synthesis by a polymerasecan be monitored by detecting hydrogen ions that are generated asnatural byproducts of nucleotide incorporations catalyzed by thepolymerase. Ion-sensitive sequencing (also referred to as “pH-based” or“ion-based” nucleic acid sequencing) exploits the direct detection ofionic byproducts, such as hydrogen ions, that are produced as abyproduct of nucleotide incorporation. In one exemplary system forion-based sequencing, the nucleic acid to be sequenced can be capturedin a microwell, and nucleotides can be flowed across the well, one at atime, under nucleotide incorporation conditions. The polymeraseincorporates the appropriate nucleotide into the growing strand, and thehydrogen ion that is released can change the pH in the solution, whichcan be detected by an ion sensor that is coupled with the well. Thistechnique does not require labeling of the nucleotides or expensiveoptical components, and allows for far more rapid completion ofsequencing runs. Examples of such ion-based nucleic acid sequencingmethods and platforms include the Ion Torrent PGM™ or Proton™ sequencer(Ion Torrent™ Systems, Life Technologies Corporation).

In some embodiments, any fragmented nucleic acids produced using themethods, systems and kits of the present teachings can be used as asubstrate for a biological or chemical reaction that is detected and/ormonitored by a sensor including a field-effect transistor (FET). Invarious embodiments the FET is a chemFET or an ISFET. A “chemFET” orchemical field-effect transistor, is a type of field effect transistorthat acts as a chemical sensor. It is the structural analog of a MOSFETtransistor, where the charge on the gate electrode is applied by achemical process. An “ISFET” or ion-sensitive field-effect transistor,is used for measuring ion concentrations in solution; when the ionconcentration (such as H+) changes, the current through the transistorwill change accordingly. A detailed theory of operation of an ISFET isgiven in “Thirty years of ISFETOLOGY: what happened in the past 30 yearsand what may happen in the next 30 years,” P. Bergveld, Sens. Actuators,88 (2003), pp. 1-20.

In some embodiments, the FET may be a FET array. As used herein, an“array” is a planar arrangement of elements such as sensors or wells.The array may be one or two dimensional. A one dimensional array can bean array having one column (or row) of elements in the first dimensionand a plurality of columns (or rows) in the second dimension. The numberof columns (or rows) in the first and second dimensions may or may notbe the same. The FET or array can comprise 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷or more FETs.

In some embodiments, one or more microfluidic structures can befabricated above the FET sensor array to provide for containment and/orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) can be configured as oneor more wells (or microwells, or reaction chambers, or reaction wells,as the terms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level,and/or concentration in the given well. In some embodiments, there canbe a 1:1 correspondence of FET sensors and reaction wells.

Microwells or reaction chambers are typically hollows or wells havingwell-defined shapes and volumes which can be manufactured into asubstrate and can be fabricated using conventional microfabricationtechniques, e.g. as disclosed in the following references: Doering andNishi, Editors, Handbook of Semiconductor Manufacturing Technology,Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMSand Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al,Silicon Micromachining (Cambridge University Press, 2004); and the like.Examples of configurations (e.g. spacing, shape and volumes) ofmicrowells or reaction chambers are disclosed in Rothberg et al, U.S.patent publication 2009/0127589; Rothberg et al, U.K. patent applicationGB24611127 (which are hereby incorporated by reference in theirentireties).

In some embodiments, the biological or chemical reaction can beperformed in a solution or a reaction chamber that is in contact with,operatively coupled, or capacitively coupled to a FET such as a chemFETor an ISFET. The FET (or chemFET or ISFET) and/or reaction chamber canbe an array of FETs or reaction chambers, respectively.

In some embodiments, a biological or chemical reaction can be carriedout in a two-dimensional array of reaction chambers, wherein eachreaction chamber can be coupled to a FET, and each reaction chamber isno greater than 10 μm³ (i.e., 1 pL) in volume. In some embodiments eachreaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pLin volume. A reaction chamber can optionally be no greater than 2, 5,10, 15, 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns incross-sectional area at the top. Preferably, the array has at least 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or more reaction chambers. In someembodiments, at least one of the reaction chambers is operativelycoupled to at least one of the FETs.

FET arrays as used in various embodiments according to the disclosurecan be fabricated according to conventional CMOS fabricationstechniques, as well as modified CMOS fabrication techniques and othersemiconductor fabrication techniques beyond those conventionallyemployed in CMOS fabrication. Additionally, various lithographytechniques can be employed as part of an array fabrication process.

Exemplary FET arrays suitable for use in the disclosed methods, as wellas microwells and attendant fluidics, and methods for manufacturingthem, are disclosed, for example, in U.S. Patent Publication No.20100301398; U.S. Patent Publication No. 20100300895; U.S. PatentPublication No. 20100300559; U.S. Patent Publication No. 20100197507,U.S. Patent Publication No. 20100137143; U.S. Patent Publication No.20090127589; and U.S. Patent Publication No. 20090026082, which areincorporated by reference in their entireties.

In one aspect, the disclosed methods, compositions, systems, apparatusesand kits can be used for carrying out label-free nucleic acidsequencing, and in particular, ion-based nucleic acid sequencing. Theconcept of label-free detection of nucleotide incorporation has beendescribed in the literature, including the following references that areincorporated by reference: Rothberg et al, U.S. patent publication2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470(2006) (which are hereby incorporated by reference in their entireties).Briefly, in nucleic acid sequencing applications, nucleotideincorporations are determined by measuring natural byproducts ofpolymerase-catalyzed extension reactions, including hydrogen ions,polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase).Examples of such ion-based nucleic acid sequencing methods and platformsinclude the Ion Torrent PGM™ or Proton™ sequencer (Ion Torrent™ Systems,Life Technologies Corporation).

In some embodiments, the disclosure relates generally to methods forsequencing nucleic acid templates. In one exemplary embodiment, thedisclosure relates generally to a method for obtaining sequenceinformation from polynucleotides, comprising: incorporating a nucleotideat the extendible end of the nucleic acid template; and detecting anon-optical signal indicating the nucleotide incorporation using asensor that detects by-products (e.g., cleavage products) from thenucleotide incorporation reaction. In some embodiments, methods forsequencing comprise: (a) providing a surface including one or morereaction sites containing a polymerase and a nucleic acid template thathas, or is hybridized to, an extendible end; (b) performing a firstnucleotide flow by contacting one or more of the reaction sites with afirst solution including one or more types of nucleotide; (c)incorporating at least one type of a nucleotide at the extendible end ofthe nucleic acid template contained within at least one of the reactionsites using the polymerase; and (d) detecting a non-optical signalindicating the nucleotide incorporation using a sensor that is attachedor operatively linked to the at least one reaction site. Optionally, thesensor comprises a FET sensor. Optionally, at least one reaction siteincludes one or more FET sensors. Optionally, the methods employ any oneor any combination of nucleotides, nucleotide analogs, and/or terminatornucleotides. Optionally, methods that employ one or more terminatornucleotides for sequencing further include: de-blocking the terminatornucleotide which is incorporated. Optionally, the methods for sequencingfurther include: performing a second nucleotide flow by contacting oneor more of the reaction sites with a second solution including one ormore types of nucleotides, where the second solution contains one ormore terminator nucleotides, one or more non-terminator nucleotides, ora mixture of both types of nucleotides. Optionally, the methods forsequencing further include: incorporating at least a second nucleotide,where the second nucleotide is a terminator nucleotide or non-terminatornucleotide from the second solution. Optionally, the methods forsequencing further include: detecting a second non-optical signalindicating the second incorporated nucleotide using the sensor that isattached or operatively linked to the at least one reaction site.

In some embodiments, the template-dependent synthesis includesincorporating one or more nucleotides in a template-dependent fashioninto a newly synthesized nucleic acid strand.

Optionally, the methods can further include producing one or more ionicbyproducts of such nucleotide incorporation.

In some embodiments, the methods can further include detecting theincorporation of the one or more nucleotides into the sequencing primer.Optionally, the detecting can include detecting the release of hydrogenions.

In another embodiment, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: disposing the nucleic acidtemplates into a plurality of reaction chambers, wherein one or more ofthe reaction chambers are in contact with a field effect transistor(FET). Optionally, the method further includes contacting the nucleicacid templates which are disposed into one of the reaction chambers,with a polymerase thereby synthesizing a new nucleic acid strand bysequentially incorporating one or more nucleotides (e.g., terminatornucleotides or non-terminator nucleotides) into a nucleic acid molecule(e.g., extendible end). Optionally, the method further includesgenerating one or more hydrogen ions as a byproduct of such nucleotideincorporation. Optionally, the method further includes detecting theincorporation of the one or more nucleotides by detecting the generationof the one or more hydrogen ions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET can be selected from the group consistingof: ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ orProton™ sequencer (Life Technologies), which is an ion-based sequencingsystem that sequences nucleic acid templates by detecting hydrogen ionsproduced as a byproduct of nucleotide incorporation. Typically, hydrogenions are released as byproducts of nucleotide incorporations occurringduring template-dependent nucleic acid synthesis by a polymerase. TheIon Torrent PGM™ or Proton™ sequencer detects the nucleotideincorporations by detecting the hydrogen ion byproducts of thenucleotide incorporations. The Ion Torrent PGM™ or Proton™ sequencer caninclude a plurality of nucleic acid templates to be sequenced, eachtemplate disposed within a respective sequencing reaction well in anarray. The wells of the array can each be coupled to at least one ionsensor that can detect the release of H⁺ ions or changes in solution pHproduced as a byproduct of nucleotide incorporation. The ion sensorcomprises a field effect transistor (FET) coupled to an ion-sensitivedetection layer that can sense the presence of H⁺ ions or changes insolution pH. The ion sensor can provide output signals indicative ofnucleotide incorporation which can be represented as voltage changeswhose magnitude correlates with the H⁺ ion concentration in a respectivewell or reaction chamber. Different nucleotide types can be flowedserially into the reaction chamber, and can be incorporated by thepolymerase into an extending primer (or polymerization site) in an orderdetermined by the sequence of the template. Each nucleotideincorporation can be accompanied by the release of H⁺ ions in thereaction well, along with a concomitant change in the localized pH. Therelease of H⁺ ions can be registered by the FET of the sensor, whichproduces signals indicating the occurrence of the nucleotideincorporation. Nucleotides that are not incorporated during a particularnucleotide flow may not produce signals. The amplitude of the signalsfrom the FET can also be correlated with the number of nucleotides of aparticular type incorporated into the extending nucleic acid moleculethereby permitting homopolymer regions to be resolved. Thus, during arun of the sequencer multiple nucleotide flows into the reaction chamberalong with incorporation monitoring across a multiplicity of wells orreaction chambers can permit the instrument to resolve the sequence ofmany nucleic acid templates simultaneously. Further details regardingthe compositions, design and operation of the Ion Torrent PGM™ orProton™ sequencer can be found, for example, in U.S. patent applicationSer. No. 12/002,781, now published as U.S. Patent Publication No.2009/0026082; U.S. patent application Ser. No. 12/474,897, now publishedas U.S. Patent Publication No. 2010/0137143; and U.S. patent applicationSer. No. 12/492,844, now published as U.S. Patent Publication No.2010/0282617, all of which applications are incorporated by referenceherein in their entireties.

In various exemplary embodiments, the methods, systems, and computerreadable media described herein may advantageously be used to processand/or analyze data and signals obtained from electronic orcharged-based nucleic acid sequencing. In electronic or charged-basedsequencing (such as, pH-based sequencing), a nucleotide incorporationevent may be determined by detecting ions (e.g., hydrogen ions) that aregenerated as natural by-products of polymerase-catalyzed nucleotideextension reactions. This may be used to sequence a sample or templatenucleic acid, which may be a fragment of a nucleic acid sequence ofinterest, for example, and which may be directly or indirectly attachedas a clonal population to a solid support, such as a particle,microparticle, bead, etc. The sample or template nucleic acid may beoperably associated to a primer and polymerase and may be subjected torepeated cycles or “flows” of nucleotide addition (which may be referredto herein as “nucleotide flows” from which nucleotide incorporations mayresult) and washing. The primer may be annealed to the sample ortemplate so that the primer's 3′ end can be extended by a polymerasewhenever nucleotides complementary to the next base in the template areadded. Then, based on the known sequence of nucleotide flows and onmeasured output signals of the chemical sensors indicative of ionconcentration during each nucleotide flow, the identity of the type,sequence and number of nucleotide(s) associated with a sample nucleicacid present in a reaction region coupled to a chemical sensor can bedetermined.

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations can be detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, templates, optionally pre-boundto a sequencing primer and/or a polymerase, can be loaded into reactionchambers (such as the microwells disclosed in Rothberg et al, citedherein), after which repeated cycles of nucleotide addition and washingcan be carried out. In some embodiments, such templates can be attachedas clonal populations to a solid support, such as particles, bead, orthe like, and said clonal populations are loaded into reaction chambers.

In another embodiment, the templates, optionally bound to a polymerase,are distributed, deposited or positioned to different sites of thearray. The site of the array include primers and the methods can includehybridizing different templates to the primers within different sites.

In each addition step of the cycle, the polymerase can extend the primerby incorporating added nucleotide only if the next base in the templateis the complement of the added nucleotide. If there is one complementarybase, there is one incorporation, if two, there are two incorporations,if three, there are three incorporations, and so on. With each suchincorporation there is a hydrogen ion released, and collectively apopulation of templates releasing hydrogen ions changes the local pH ofthe reaction chamber. The production of hydrogen ions is monotonicallyrelated to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, can be proportional tothe number of contiguous identical complementary bases. If the next basein the template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepcan be performed, in which an unbuffered wash solution at apredetermined pH is used to remove the nucleotide of the previous stepin order to prevent misincorporations in later cycles. In someembodiments, the after each step of adding a nucleotide, an additionalstep can be performed wherein the reaction chambers are treated with anucleotide-destroying agent, such as apyrase, to eliminate any residualnucleotides remaining in the chamber, which may result in spuriousextensions in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction can beexposed to the different nucleotides one at a time. For example,nucleotides can be added in the following sequence: dATP, dCTP, dGTP,dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed bya wash step. The cycles may be repeated for 50 times, 100 times, 200times, 300 times, 400 times, 500 times, 750 times, or more, depending onthe length of sequence information desired.

In some embodiments, sequencing can be performed according to the userprotocols supplied with the PGM™ or Proton™ sequencer. Example 3provides one exemplary protocol for ion-based sequencing using the IonTorrent PGM™ sequencer (Ion Torrent™ Systems, Life Technologies, CA).

In some embodiments, the disclosure relates generally to methods forsequencing a population of template polynucleotides, comprising: (a)generating a plurality of amplicons by clonally amplifying a pluralityof template polynucleotides onto a plurality of surfaces, wherein theamplifying is performed within a single continuous phase of a reactionmixture and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% of the resulting amplicons are substantially monoclonal innature. In some embodiments, a sufficient number of substantiallymonoclonal amplicons are produced in a single amplification reaction togenerate at least 100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1 GBor 2 GB of AQ20 sequencing reads on an Ion Torrent PGM™ 314, 316 or 318sequencer. The term “AQ20 and its variants, as used herein, refers to aparticular method of measuring sequencing accuracy in the Ion TorrentPGM™ sequencer. Accuracy can be measured in terms of the Phred-like Qscore, which measures accuracy on logarithmic scale that: Q10=90%,Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. For example, in aparticular sequencing reaction, accuracy metrics can be calculatedeither through prediction algorithms or through actual alignment to aknown reference genome. Predicted quality scores (“Q scores”) can bederived from algorithms that look at the inherent properties of theinput signal and make fairly accurate estimates regarding if a givensingle base included in the sequencing “read” will align. In someembodiments, such predicted quality scores can be useful to filter andremove lower quality reads prior to downstream alignment. In someembodiments, the accuracy can be reported in terms of a Phred-like Qscore that measures accuracy on logarithmic scale such that: Q10=90%,Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. In someembodiments, the data obtained from a given polymerase reaction can befiltered to measure only polymerase reads measuring “N” nucleotides orlonger and having a Q score that passes a certain threshold, e.g., Q10,Q17, Q100 (referred to herein as the “NQ17” score). For example, the100Q20 score can indicate the number of reads obtained from a givenreaction that are at least 100 nucleotides in length and have Q scoresof Q20 (99%) or greater. Similarly, the 200Q20 score can indicate thenumber of reads that are at least 200 nucleotides in length and have Qscores of Q20 (99%) or greater.

In some embodiments, the accuracy can also be calculated based on properalignment using a reference genomic sequence, referred to herein as the“raw” accuracy. This is single pass accuracy, involving measurement ofthe “true” per base error associated with a single read, as opposed toconsensus accuracy, which measures the error rate from the consensussequence which is the result of multiple reads. Raw accuracymeasurements can be reported in terms of “AQ” scores (for alignedquality). In some embodiments, the data obtained from a given polymerasereaction can be filtered to measure only polymerase reads measuring “N”nucleotides or longer having a AQ score that passes a certain threshold,e.g., AQ10, AQ17, AQ100 (referred to herein as the “NAQ17” score). Forexample, the 100AQ20 score can indicate the number of reads obtainedfrom a given polymerase reaction that are at least 100 nucleotides inlength and have AQ scores of AQ20 (99%) or greater. Similarly, the200AQ20 score can indicate the number of reads that are at least 200nucleotides in length and have AQ scores of AQ20 (99%) or greater.

EXAMPLES

Examples of enzymatic compositions and methods are directed tocontrolled in vitro fragmentation of nucleic acids, generation of DNAfragments containing shortened stretches of transposon end sequences,and production of asymmetrically tailed DNA fragments.

Materials and Methods

All enzymes, except stand-alone MuA transposase, and reagents were fromMuSeek Library Preparation Kit for Ion Torrent (Cat. No. K1331, ThermoScientific) unless indicated otherwise.

Stand-alone MuA transposase enzyme was from Thermo Scientific (Cat. No.F-750). All oligonucleotides were synthesized at Metabion.

Transposon ends for Examples 1-3 at a final concentration of 60 μM wereprepared by annealing equimolar quantities of primers in annealingbuffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl):

Cut-key4 (No_nick) and Non-cut-key4 or

Cut-key4 (1Nick45-5), Cut-key4 (2Nick45-5) and Non-cut-key4 or

Cut-key4 (1Nick44-6), Cut-key4 (2Nick44-6) and Non-cut-key4 or

Cut-key4 (1Nick42-8), Cut-key4 (2Nick42-8) and Non-cut-key4 or

Cut-key4 (1Nick40-10), Cut-key4 (2Nick40-10) and Non-cut-key4 or

Cut-key4 (1Nick38-12), Cut-key4 (2Nick38-12) and Non-cut-key4 or

Cut-key4 (1Nick36-14), Cut-key4 (2Nick36-14) and Non-cut-key4 or

Cut-key4 (1Nick34-16), Cut-key4 (2Nick34-16) and Non-cut-key4 or

Cut-key4 (1Nick32-18), Cut-key4 (2Nick32-18) and Non-cut-key4 or

Cut-key4 (1Nick31-19), Cut-key4 (2Nick31-19) and Non-cut-key4 or

Cut-key4 (1Nick23-27), Cut-key4 (2Nick23-27) and Non-cut-key4

Transposon ends for Examples 4-6 at a final concentration of 40 μM wereprepared by annealing equimolar quantities of primers in annealingbuffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl):

Cut-key4 (No_nick) and Non-cut-key4 or

Cut-key4 (1Nick 38-12), Cut-key4 (2Nick 38-12) and Non-cut-key4 or

Cut-key4 (1Gap 42-6), Cut-key4 (2Gap 42-6) and Non-cut-key4 or

Cut-key4 (1Gap 40-8), Cut-key4 (2Gap 40-8) and Non-cut-key4 or

Annealing of oligonucleotides was accomplished by using the PCRinstrument Eppendorf Mastercycler ep Gradient S (Eppendorf) and thefollowing program: 95° C. for 5 min, 70 cycles each lasting for 40seconds and gradually decreasing the temperature of the block by 1° C.at the end of each cycle starting from 95° C. and ending with 25° C.,followed by incubation at 10° C. until the annealed oligonucleotideswere used for complex formation. All oligonucleotide sequences used fortransposon end preparation are shown in Table 1 and Table 2.

Table 1 below shows the sequences of oligonucleotides used to formvarious pre-nicked transposon ends or full length transposon ends aswell as sequences of oligonucleotides used for DNA amplification.

No. Title Oligonucleotide sequence  1 Non-cut-key4GCGAAAGCGTTTCACGATAAATGCGAAAAC  2 Cut-key4GTTTTCGCATTTATCGTGAAACGCTTTCGC (No_nick) GTTTTTCGTGCGTCAGTTCA  3Cut-key4 AGTTTTCGCATTTATCGTGAAACGCTTTCG (1 Nick45-5) CGTTTTTCGTGCGTC  4Cut-key4 GTTCA (2 Nick45-5)  5 Cut-key4 GTTTTCGCATTTATCGTGAAACGCTTTCGC(1 Nick44-6) GTTTTTCGTGCGTC  6 Cut-key4 AGTTCA (2 Nick44-6)  7 Cut-key4GTTTTCGCATTTATCGTGAAACGCTTTCGC (1 Nick42-8) GTTTTTCGTGCG  8 Cut-key4TCAGTTCA (2 Nick42-8)  9 Cut-key4 GTTTTCGCATTTATCGTGAAACGCTTTCGC(1 Nick40-10) GTTTTTCGTG 10 Cut-key4 CGTCAGTTCA (2 Nick40-10) 11Cut-key4 GTTTTCGCATTTATCGTGAAACGCTTTCGC (1 Nick38-12) GTTTTTCG 12Cut-key4 TGCGTCAGTTCA (2 Nick38-12) 13 Cut-key4GTTTTCGCATTTATCGTGAAACGCTTTCGC (1 Nick36-14) GTTTTT 14 Cut-key4CGTGCGTCAGTTCA (2 Nick36-14) 15 Cut-key4 GTTTTCGCATTTATCGTGAAACGCTTTCGC(1 Nick34-16) GTTT 16 Cut-key4 TTCGTGCGTCAGTTCA (2 Nick34-16) 17Cut-key4 GTTTTCGCATTTATCGTGAAACGCTTTCGC (1 Nick32-18) GT 18 Cut-key4TTTTCGTGCGTCAGTTCA (2 Nick32-18) 19 Cut-key4GTTTTCGCATTTATCGTGAAACGCTTTCGC (1 Nick31-19) G 20 Cut-key4TTTTTCGTGCGTCAGTTCA (2 Nick31-19) 21 Cut-key4 GTTTTCGCATTTATCGTGAAACG(1 Nick23-27) 22 Cut-key4 CTTTCGCGTTTTTCGTGCGTCAGTTCA (2 Nick23-27) 23Primer A CCATCTCATCCCTGCGTGTCTTCGTGCGTC AGTTCA 24 Primer P1CCACTACGCCTCCGCTTTCCTCTCTATGGG CAGTCGGTGATTTCGTGCGTCAGTTCA 25 Primer A′CCATCTCATCCCTGCGTGTC 26 Primer P1′ CCACTACGCCTCCGCTTTCCTCTCTATG

Table 2 below shows the sequences of oligonucleotides used to form fulllength (native) transposon ends, pre-nicked transposon ends andtransposon ends with gaps for DNA fragmentation experiments using MuAtranspososome containing nicked and gapped transposon ends (Examples4-6).

Oligonucleotide Purpose No. Title sequence of use 1 Non-cut-key4ACGACTTGACTGCGT All GCTTTTTGCGCTTTC transposons GCAAAGTGCTATTTA CGC 2Cut-key4 GTTTTCGCATTTATC Native (No_nick) GTGAAACGCTTTCGC transposonGTTTTTCGTGCGTCA GTT 3 Cut-key4 GTTTTCGCATTTATC Nick 38-12 (1Nick 38-12)GTGAAACGCTTTCGC transposon GTTTTTCG 4 Cut-key4 TGCGTCAGTTCA Nick 38-12(2Nick 38-12) transposon 5 Cut-key4 GTTTTCGCATTTATC Gap 42-6 (1Gap 42-6)GTGAAACGCTTTCGC transposon GTTTTTCGTGCG 6 Cut-key4 AGTTCA Gap 42-6(2Gap 42-6) transposon 7 Cut-key4 GTTTTCGCATTTATC Gap 40-8 (1Gap 40-8)GTGAAACGCTTTCGC transposon GTTTTTCGTG 8 Cut-key4 TCAGTTCA Gap 40-8(2Gap 40-8) transposon

FIG. 8 shows the structure of transposon ends designed for experimentsin Examples 4-6.

Complex Assembly Buffer is 150 mM Tris-HCl pH 6.0, 50% (v/v) glycerol,0.025% (w/v) Triton X-100, 150 mM NaCl, 0.1 mM EDTA.

Extra DMSO is 4.6% DMSO at final concentration.

Fragmentation Reaction Buffer is MuSeek Fragmentation Reaction Buffer(Thermo Scientific MuSeek Library Preparation Kit for Ion Torrent™,#K1331) or alternatively 36 mM Tris-HCl (pH 8.0), 137 mM NaCl, 0.05%Triton X-100, 10 mM MgCl₂, 4.6% DMSO, and 6.8% glycerol.

Dilution Buffer is 47.2% glycerol, 200 mM NaCl, and 2 mM EDTA at finalconcentrations

Example 1 MuA Transposase Forms Catalytically Active Complexes withPre-Nicked Transposon Ends

The ability of MuA transposase to form catalytically active complexeswith pre-nicked transposon ends was directly shown by fragmenting E.coli genomic DNA using MuA transpososomes formed with several differentpre-nicked transposon ends.

MuA transposomes were formed in Complex Assembly Buffer with extra DMSO.Final concentration of transposon end was 8 μM and for MuA transposase1.65 g/l in complex assembly reaction (this is equimolar concentrationfor MuA transposome formation). After one hour incubation at 30° C., thecomplex assembly mix was diluted with Dilution Buffer. The final dilutedMuA transposome complex concentration was about 0.48 g/l. MuAtransposome complex was stored at −70° C. for at least 16 hours beforeuse.

Escherichia coli str. K-12 substr. DH10B gDNA was fragmented usingtranspososomes made from 10 different pre-nicked transposon ends and onefull length (native) transposon end. Each of eleven fragmentationreactions was carried out in separate tube using 100 ng E. coli gDNA inFragmentation Reaction Buffer. Immediately after adding the transposomemix (1 μl to final reaction volume 30 μl), vortexing, and a shortspin-down, the tube was incubated at 30° C. for five minutes. Thefragmentation reaction was stopped by adding 3 μl 4.4% SDS. After briefvortexing, the tube was kept at room temperature.

Fragmented DNA was purified by Agencourt AMPure XP PCR Purification(Beckman Coulter) system as follows. Fragmented DNA was transferred intoa 1.5 ml tube. Then 49.5 μl of thoroughly mixed Agencourt AMPure XP(Beckman Coulter) beads were added to the reaction and mixed carefullyby pipetting up and down ten times. The same procedure was applied toall eleven samples of fragmented DNA. Samples were incubated for fiveminutes at room temperature. After a short spin, the tubes were placedin a magnetic rack until the solutions were cleared. The supernatant wasaspirated carefully without disturbing the beads and discarded. Thetubes were kept in the magnetic rack and 400 μl of freshly prepared 70%ethanol was added. After 30 seconds of incubation the supernatant wasremoved. The ethanol wash step was repeated. The beads were thenair-dried by opening the tube caps for two minutes, allowing remainingethanol to evaporate. The tubes were removed from the magnetic rack, andthe beads were suspended in 25 μl of nuclease free water by pipetting upand down ten times. The tubes were then placed back in the magnetic rackand after the solution became clear all 11 supernatants containing thepurified fragmented DNA were carefully transferred to new sterile tubes.

The purified fragmented DNAs were analyzed using the Agilent 2100Bioanalyzer (Agilent Biotechnologies) and the Agilent High SensitivityDNA Kit (Agilent Biotechnologies) following manufacturer'srecommendations. Before analysis, fragmented DNA was diluted by addingequal volume of nuclease free water.

Analysis of fragmentation reaction products (FIG. 2) revealed shorteningof gDNA in all cases indicating that transposon ends comprising a nickin general do not interfere with transposase catalytic activity. Invitro transposition reactions resulted in DNA fragments ranging fromabout 100 bp to 2000 bp in length. Similar curves of DNA fragment lengthdistribution were observed when standard full length transposon end(i.e. native transposon end used for in vitro transposition reactioncontrol) and transposon ends containing nicks after the 6^(th), 8^(th),10^(th), 14^(th), 16^(th), 18^(th), 19^(th), 27^(th) positions from the3′ end of Cut-key4 oligonucleotide were used. However, nicks introducedafter 5^(th) and 12^(th) positions from the 3′ end of Cut-key4oligonucleotide reduced DNA fragmentation reaction efficiency.

Example 2 MuA Transposase in Combination with Pre-Nicked Transposon Endscan be Used as a Universal and Well-Controlled DNA Fragmentation ToolSuitable for NGS and Other Molecular Biology Downstream Applicationswhich Require DNA Shearing

Versatility of MuA transposase/pre-nicked transposon end fragmentationmethod was demonstrated by fragmenting various DNA substrates: highcomplexity human gDNA, double stranded copy DNA made from mRNA isolatedfrom HeLa cells, and three microbial gDNAs differing significantly intheir GC content: 50% GC (Escherichia coli str. K-12 substr. DH10B),high GC (Thermus thermophilus str. HB8) and high AT (Staphylococcusaureus str. Mu50). Well-controlled fragmentation was demonstrated byshearing E. coli gDNA using varying amounts of MuAtransposase-pre-nicked transposon end complex that resulted in distinct,transposome amount dependent average length of DNA fragments.Suitability of DNA fragments resulting after the shearing of DNA ofinterest with nick containing transposon ends for next generationsequencing was demonstrated using sheared E. coli gDNA to prepare IonTorrent PGM (Life Technologies) compatible DNA library, which wassubsequently sequenced to demonstrate run summary segments whichindicated good DNA library quality.

Transposon ends (final concentration 60 μM) were prepared by annealingequimolar quantities of primers Cut-key4 (1Nick44-6), Cut-key4(2Nick44-6) and Non-cut-key4 in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50mM NaCl and following conditions described in Materials and Methodsabove.

MuA transposomes were formed in complex assembly buffer with DMSO. Thefinal concentration of transposon end was 8 μM and for MuA transposase1.65 g/l in complex assembly reaction (this is equimolar concentrationfor MuA transposome formation). After one hour incubation at 30° C.,complex assembly mix was diluted with Dilution Buffer. Final diluted MuAtransposome complex concentration was about 0.48 g/l. MuA transposomecomplex was stored at −70° C. for at least 16 hours before use.

HeLa double stranded copy DNA was synthesized from 500 ng of HeLa mRNAusing Maxima H minus Double-Stranded cDNA Synthesis Kit (ThermoScientific) according to the manufacturer's protocol. After ds cDNAsynthesis, the sample was purified using GeneJET PCR Purification Kit(Thermo Scientific) following manufacturer recommendations.Double-stranded cDNA was eluted with 20 μL of elution buffer.

Human gDNA, HeLA double-stranded cDNA, Escherichia coli str. K-12substr. DH10B gDNA, Thermus thermophilus str. HB8 and Staphylococcusaureus str. Mu50 gDNA were fragmented using the 44-6 pre-nickedtransposon end containing MuA transpososome. Fragmentation reactionswere carried out in separate tubes using 100 ng of any DNA infragmentation reaction buffer. Immediately after adding the transposomemix (1 μl to the final reaction volume of 30 μl, or in case ofcontrolled E. coli gDNA fragmentation 0.25 μl, 0.5 μl, 1.5 μl and 2 μlto the final reaction volume of 30 μl), vortexing and a short spin-down,the tubes were incubated at 30° C. for five minutes. Subsequently,fragmentation reactions were stopped by adding 3 μl 4.4% SDS. Afterbrief vortexing, tubes were kept at room temperature.

Fragmented DNAs were purified using Agencourt AMPure XP PCR Purification(Beckman Coulter) system. Fragmented DNAs were transferred into 1.5 mltubes. Then 49.5 μl of thoroughly mixed AMPure XP (Beckman Coulter)beads were added to reaction mixtures and mixed carefully by pipettingup and down for ten times. Samples were incubated for five minutes atroom temperature. After a short spin, the tubes were placed in amagnetic rack until the solutions were cleared. The supernatant wasaspirated carefully without disturbing the beads and discarded. Thetubes were kept in the rack and 400 μl of freshly prepared 70% ethanolwas added. After 30 seconds of incubation supernatant was carefullyremoved, and the same washing procedure repeated. The beads were thenair-dried for two minutes, tubes removed from the magnetic rack, andbeads resuspended in 25 μl of nuclease-free water by pipetting up anddown ten times. The tubes were placed back in the magnetic rack untilthe solution became clear, and supernatants containing the elutedpurified fragmented DNA were transferred to new tubes.

The purified fragmented DNAs were analyzed using the Agilent 2100Bioanalyzer (Agilent Biotechnologies) and the Agilent High SensitivityDNA Kit (Agilent Biotechnologies) following manufacturer'srecommendations. Before analysis, fragmented DNA was diluted by addingequal volume of nuclease free water.

Intact E. coli gDNA, 1.5 ng, was analyzed on Agilent 2100 Bioanalyzer(Agilent Biotechnologies) as well and served as an uncleaved control.

A library of DNA fragments ready for sequencing on Ion Torrent PGM (LifeTechnologies) instrument was prepared from 100 ng of E. coli gDNAfragmented with 1.5 μl of MuA transpososome made from pre-nickedtransposon end 44-6. Ends of purified DNA fragments were polished andIon Torrent-compatible adaptors were ligated using ClaSeek LibraryPreparation Kit (Thermo Scientific) according to the manufacturer'sprotocol. After adaptor ligation, reaction products were purified andsize-selected for 200 bp sequencing using MagJet NGS Cleanup and SizeSelection Kit (Thermo Scientific). Size-distribution of resulting DNAfragments before and after adapter ligation was analyzed on Agilent 2100Bioanalyzer (Agilent Biotechnologies) with results shown in FIG. 5. TheDNA library was quantified using Ion Library Quantitation Kit (LifeTechnologies), sequencing template was prepared using Ion PGM TemplateOT2 200 Kit (Life Technologies) and subsequently sequenced on IonTorrent PGM (Life Technologies) using Ion PGM 200 Sequencing Kit (LifeTechnologies) according to the manufacturer's protocol. The sequencingreport was generated with TorrentSuite (Life Technologies) software,version 3.6.

Feasibility of MuA transposase usage in conjunction with pre-nicked 44-6transposon end as a universal and controlled DNA fragmentation tool wasdemonstrated with various DNA substrates. Five DNA samples were tested:high complexity human gDNA, double stranded copy DNA made from HeLamRNA, Staphylococcus aureus str. Mu50 gDNA featuring low GC content(33%), Escherichia coli str. K-12 substr. DH10B gDNA which GC content is51%, and Thermus thermophilus str. HB8 gDNA (GC content 69%). In allcases fragmentation reactions yielded DNA fragments ranging in lengthfrom 100 bp to 2000 bp, as shown in FIG. 3. The ability to adjust thelength of shared DNA fragments was demonstrated in E. coli gDNAfragmentation experiments. The results indicated that DNA fragmentationlevel was inversely dependent on the amount of MuA-transposome complexused: adding 0.25 μl cleaved genomic DNA to fragments of 1850 bp onaverage, 0.5 μl-˜800 bp, 1.5 μl-˜450 bp, 2 μl-˜400 bp shown in FIG. 4.Those skilled in the art will understand that the level of DNA shearingmay be manipulated by changing the amount of added transpososome andalso by either reducing or increasing the amount of input DNA, and thatthe level of sharing depends on the integrity of input DNA: in order toachieve the same DNA fragmentation level of very large gDNA fragmentsone needs to add more transpososome compared to the partially degradedlow-quality DNA sample. Sequencing results of E. coli gDNA libraryprepared by using 1.5 μl of transpososome made from the pre-nickedtransposon end 44-6 (FIG. 6) revealed good DNA library quality (i.e.small amount of low quality reads, high percentage of aligned reads,high mean raw accuracy), clearly indicating that the DNA shearingapproach which exploits pre-nicked transposon ends may be used toprepare high quality DNA libraries for NGS. It is important to note thatthe pre-nicked transposon end 44-6 used for feasibility studies containsa nick after the 6^(th) position from the 3′ end of Cut-key4oligonucleotide and thus leaves 6 nucleotides at each end of shared DNAwhich originate from the transposon end. For that reason transposon endsremaining after the polishing of shared DNA fragment ends were trimmedout together with adaptor sequences during sequencing data analysis.This explains why mean sequencing read length (FIG. 6) was ˜100 bpshorter than the DNA library used for sequencing (FIG. 5, curve B).Those skilled in the art will understand that the blunt-ended ligationof adapters to polished DNA fragment ends can be easily replaced bysticky-end adapter ligation using specially designed adapters featuringsticky ends which are complementary to those generated by DNAfragmentation with a transpososome made with pre-nicked transposon end.

Example 3 The Use of MuA Transposase in Combination with Pre-NickedTransposon Ends for DNA Fragmentation Generated DNA Fragments that wereAmplified More Efficiently

DNA fragments generated using MuA transpososome containing 34-16pre-nicked transposon end had improved PCR efficiency. This wasdemonstrated by comparing the outcome of PCR of human gDNA fragmentedwith the MuA transpososome containing the stated nicked transposon endand the MuA transpososome containing the standard full-length transposonend.

Transposon ends (final concentration 60 μM) were prepared by annealingequimolar quantities of primers Cut-key4 (1Nick34-16), Cut-key4(2Nick34-16) and Non-cut-key4 or Cut-key4 (No_nick) and Non-cut-key4 in10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl, following the conditionsdescribed in Materials and Methods.

MuA transposomes were formed in complex assembly buffer with extra DMSO.The final concentration of transposon end was 8 μM and for MuAtransposase 1.65 g/l in the complex assembly reaction. After one hourincubation at 30° C., the complex assembly mix was diluted with dilutionbuffer. Final diluted MuA transposome complex concentration was about0.48 g/l. MuA transposome complex was stored at −70° C. for at least 16hours before use.

Human gDNA was fragmented in parallel with MuA transpososome containingeither 34-16 pre-nicked transposon end or a standard full lengthtransposon end. Reactions were performed using 100 ng human DNA infragmentation reaction buffer. Immediately after adding the transposomemix (1 μl to final reaction volume 30 μl), vortexing and a shortspin-down, the tube was incubated at 30° C. for five minutes. Thereaction was stopped by adding 3 μl 4.4% SDS. After brief vortexing, thetube was kept at room temperature.

Fragmented DNA was purified using the Agencourt AMPure XP PCRPurification (Beckman Coulter) system following the protocol describedin Example 1.

Purified fragmented human gDNAs served as templates for PCRamplification with Phusion Hot Start II High-Fidelity DNA polymerase(Thermo Scientific) and 4 primers: A and P1-Ion platform specificadaptors with transposon end sequences, A′ and P1′-PCR amplificationprimers.

A 5′-CCATCTCATCCCTGCGTGTCTTCGTGCGTCAGTTCA-3′, P15′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATTT CGTGCGTCAGTTCA-3′, A′5′-CCATCTCATCCCTGCGTGTC-3′, P1′ 5′-CCACTACGCCTCCGCTTTCCTCTCTATG-3′.

Reactions were carried out using 20 μl of fragmented DNA (in finalvolume of 200 μl) in 10 mM Tris-HCl, pH 8.8, 110 mM KCl, 1.5 mM MgCl₂,0.1% (w/v), Triton X-100, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 200 μMdGTP using the following cycling conditions: 66° C. 3′, 1×98° C. 30″;9×98° C. 10″, 60° C. 50″, 72° C. 10″; 1×72° C. 1′.

Amplified DNA was purified using the Agencourt AMPure XP PCRPurification (Beckman Coulter) system. Fragmented DNA was transferredinto a 1.5 ml tube. Then 360 μl of thoroughly mixed Agencourt AMPure XP(Beckman Coulter) beads were added to the reaction and mixed carefullyby pipetting up and down ten times. Samples were incubated for fiveminutes at room temperature. After a short spin, the tubes were placedin a magnetic rack until the solutions were cleared. The supernatant wasaspirated carefully without bead disruption and discarded. The tubeswere kept in the rack and 1000 μl of freshly prepared 70% ethanol wasadded. After 30 seconds of incubation supernatant was removed. Theethanol wash step was repeated. The beads were air-dried, the tubesremoved from the magnetic rack, and the beads were suspended in 20 μl ofnuclease-free water by pipetting up and down ten times. The tubes werethen placed in the magnetic rack until the solution became clear andsupernatants containing the eluted DNA were transferred into new tubes.

DNA samples before and after PCR amplification were analyzed using theAgilent 2100 Bioanalyzer (Agilent Biotechnologies) and the Agilent HighSensitivity DNA Kit (Agilent Biotechnologies). Before analysis,fragmented DNA was diluted with nuclease free water by a factor of 2,while amplified samples by a factor of 4.

The results are shown in FIG. 7. They revealed differences in PCRamplification outcome among DNA samples analyzed. DNA samples fragmentedwith MuA transpososome containing the transposon ends having a nickafter 16^(th) position (from the 3′ end) of Cut-key4 oligonucleotideresulted in reduced amount of short DNA fragments that are unwanted inNGS, thus significantly improving the quality of PCR-amplified DNAlibrary.

Example 4 MuA Transposase Forms Catalytically Active Complexes withTransposon Ends Containing Gap

The ability of MuA transposase to form catalytically active complexeswith transposon ends containing nucleotide gaps was demonstrated byfragmenting E. coli genomic DNA using MuA transpososomes formed withseveral different transposon ends harboring gap.

Transposon ends at a final concentration of 40 μM were prepared asdescribed in Materials and Methods.

MuA-transposon end complexes (transposon mixes) were formed in complexassembly buffer with extra DMSO. The final concentration of transposonend was 8 μM and for MuA transposase 1.65 g/l in complex assemblyreaction (this is equimolar concentration for MuA transposomeformation). After one hour incubation at 30° C., complex assembly mixwas diluted with dilution buffer. Final diluted MuA-transposon endcomplex concentration was about 0.48 g/l. MuA-transposon end complex wasstored at −70° C. for at least 16 hours before use.

Escherichia coli str. K-12 substrate DH10B gDNA was fragmented using MuAtranspososomes made from 38-12 pre-nicked transposon end, 42-6transposon end with gap and 40-8 transposon end with gap or full lengthnative transposon end for use as a control of in vitro transpositionreaction, abbreviated in the figures as “MuSeek”. Each of fourfragmentation reactions was carried out in a separate tube using 100 ngE. coli gDNA in fragmentation reaction buffer. Immediately after addingMuA-transposon end complexes (1.5 μl to final reaction volume 30 μl),vortexing and a short spin-down, the tube was incubated at 30° C. forfive minutes. The fragmentation reaction was stopped by adding 3 μl of4.4% SDS. After brief vortexing, the tube was kept at room temperature.Fragmented DNA was purified using GeneJET NGS Purification Kit (ThermoScientific) and analyzed using Agilent 2100 Bioanalyzer (AgilentBiotechnologies) and Agilent High Sensitivity DNA Kit (AgilentBiotechnologies).

Results, shown in FIG. 9, demonstrated that MuA transposase formedhighly catalytically active complexes with full length native transposonends, abbreviated in the figure as “MuSeek”, resulting in random DNAfragments distributed over a broad range of length. Transposon ends withnucleotide gaps (Gap42-6, Gap 40-8) also formed catalytically activecomplex with MuA transposase and were able to fragment DNA. DNAfragmentation profile of MuA transposase-gapped transposon end complexeswas similar to previous DNA fragmentation experiments using MuAtransposase-nicked (38-12) transposon end complexes.

Example 5 MuA Transpososomes Containing Transposon Ends with Gaps can beUsed as a Controlled DNA Fragmentation Tool Enabling Generation of DNAFragments Having Predefined Average Length

DNA fragment length dependence on the transposon end structure assembledinto MuA transpososome, amount of MuA transpososome, and in vitrotransposition reaction time was shown by fragmenting E. coli genomic DNAusing MuA transpososomes formed with native transposon ends, nickedtransposon ends, or transposon ends having nucleotide gaps.

Transposon ends at a final concentration of 40 μM were prepared asdescribed in Materials and Methods.

MuA-transposon end complexes (transposome mixes) were formed in complexassembly buffer with extra DMSO. The final concentration of transposonend was 8 μM and for MuA transposase 1.65 g/l in complex assemblyreaction (this is equimolar concentration for MuA transposomeformation). After one hour incubation at 30° C., complex assembly mixwas diluted with dilution buffer. Final diluted MuA-transposon endcomplex concentration was about 0.48 g/l. MuA-transposome complex wasstored at −70° C. for at least 16 hours before use.

Escherichia coli str. K-12 substr. DH10B gDNA was fragmented using MuAtranspososomes made from 38-12 pre-nicked transposon ends, 42-6transposon ends with gap or full length native transposon ends used ascontrol of in vitro transposition reaction, abbreviated in the figuresas “MuSeek”. Each fragmentation reaction was carried out in separatetube incubating at 30° C. 100 ng of E. coli gDNA in fragmentationreaction buffer with different amount (0.5 μl and 1.5 μl in finalreaction volume 30 μl) of MuA-transposome complex for different periodsof time of 1.5 minutes, 5 minutes, and 10 minutes, respectively. Thefragmentation reaction was stopped by adding 3 μl of 4.4% SDS. Afterbrief vortexing, the tube was kept at room temperature. Fragmented DNAwas purified using GeneJET NGS Purification Kit (Thermo Scientific) andanalyzed using Agilent 2100 Bioanalyzer (Agilent Biotechnologies) andAgilent High Sensitivity DNA Kit (Agilent Biotechnologies).

Results, shown in FIG. 10, indicated that DNA fragmentation using 1.5 μlof MuA-native transposon end complex at in vitro transposition reactiontime of 1.5 min-10 min resulted in random DNA fragments distributed overa broad range of length from about 200 bp to 2000 bp. DNA fragmentationusing the same or lower amount of MuA-nicked transposon end (Nick 38-12)complex or MuA-transposon end with gap (Gap 42-6) complex at in vitrotransposition reaction of 1.5 min-10 min resulted in specific DNAfragmentation profile, where only DNA fragments of predefined lengthwere generated distributed from about 1 kb to 6 kb. These resultssupport the assumption that DNA fragmentation reaction catalyzed by MuAtranspososomes where transposon ends are modified to contain gaps ornicks proceeds slower compared to DNA fragmentation reaction catalyzedby MuA-native transposon end complex. Subsequently, the efficiency of invitro transposition reaction was investigated using lower DNA input andprolonged incubation time. Amounts of 25 ng, 50 ng and 100 ng of E. coligDNA were incubated with 1.5 μl of MuA-native transposon end complex andMuA-transposon end with gap (Gap 42-6) complex for 30 min. FragmentedDNA was purified and analyzed as indicated above. Results, shown in FIG.11, demonstrated that lower amount of DNA input in the fragmentationreaction mixture and prolonged incubation time resulted in the shift ofDNA fragmentation reaction products towards shorter length for bothMuA-native transposon end complex and MuA-transposon end with gap (Gap42-6) complex. These results revealed that the length of DNAfragmentation reaction products could be predefined by manipulating theamount of catalytically active MuA-transposon end complex, in vitrotransposition reaction time, or DNA input amount. For example, similarDNA fragmentation profiles were achieved for the DNA fragmentationreaction where the amount of input DNA (100 ng) and MuA transpososome (3μl) containing either native transposon end or 42-6 transposon end withgap was the same by prolonging DNA fragmentation reaction time up to 10min catalyzed by MuA transpososome containing 42-6 transposon end withgap (FIG. 12).

The examples support the novel teaching of this disclosure, which isbased on the observation that transpososomes where transposon ends aremodified to contain gaps, nicks, or apurinic/apyrimidinic sites retainedtheir ability to enzymatically shear various DNA substrates, and thatgaps, nicks, or apurinic/apyrimidinic sites within the transposon endslowed in vitro transposition reaction rate catalyzed by transposaseenzyme. As a result, DNA fragmentation reaction kinetics (i.e.transposase-transposon end complex amount, DNA input amount, and/or DNAfragmentation reaction time) can be adjusted such that only DNAfragments of predefined average length are generated providing foruniversal and controlled DNA fragmentation.

Example 6 MuA Transposase Forms Catalytically Active Complexes withModified Transposon Ends Containing Degenerate Sequences and can be Usedas a Controlled DNA Fragmentation Tool Enabling Generation of DNAFragments Having Predefined Average Length

A number of transposon end sequences that differ substantially from thewild-type MuA transposon sequence (native sequence) were analyzed fortheir ability to participate in a transposition reaction. A transposonlibrary comprising transposon ends with degenerated nucleotidesintroduced in locations that, based on scientific literature arereferred to as conserved sites, was prepared and used for thetransposition reaction catalyzed by MuA. Resulting transpositionproducts were sequenced and analyzed in terms of sequence variation.

Results are shown in FIG. 13. In this experiment degenerated nucleotidemixtures were prepared so each N represents a special wobble mixture,where nucleotide found in the wild-type transposon sequence was retainedwith 70% frequency, while other three nucleotides were inserted withequal frequencies of 10%:10%:10%. Number 1 on vertical axis represents100% congruence between expected nucleotide distribution frequency(70%:10%:10%:10%) and factual frequency calculated from sequencing data.The smaller the number on vertical axis, the higher deviation fromexpected nucleotide distribution frequency was observed at a certainposition. For simplicity only the transferred DNA strand is shown.

Not all the conserved positions were found to be of high importance forthe transposition efficiency in the experimental framework. However, MuAtransposase has some preferences towards a number of positions in atransposon. Thus, the skilled person in the art may design differenttransposons for which MuA transposase has lower affinity. Afterwards,several modified transposons that were still able to supporttransposition reaction, albeit with lower efficiency, were selected andanalyzed.

Several different transposons selected in the first experiment ascapable of supporting transposition (Table 3) were used for transposomecomplex formation with MuA transposase in equimolar concentrations asdescribed above. The final concentration of transposon DNA was 8 μM andfor MuA transposase 1.65 g/l in complex assembly reaction. After onehour incubation at 30° C., complex assembly mix was diluted withDilution Buffer. The final diluted MuA transposome complex concentrationwas about 0.48 g/l. MuA transposome complex was stored at −70° C. for atleast 16 hours before use. Subsequently, E. coli genomic DNA wassubjected to the fragmentation reaction employing the pre-formedcomplexes. Fragmentation reaction products were purified by AgencourtAMPure XP system (Beckman Coulter) and analyzed on Agilent 2100Bioanalyzer using Agilent High Sensitivity DNA Kit (AgilentBiotechnologies) following manufacturer's recommendations. FIG. 14 showsthat gDNA fragmentation was very efficient when Control transposon(native transposon end sequence) was used, while for modified transposonends fragmentation was apparent only with transposon Nos. 5, 10, and 11.Fragmentation was completely abolished when transposon No. 9 was used.

In parallel, EMSA analysis was performed for all pre-formedMuA-transposon complexes used in the first experiment (Table 3). EMSAanalysis results are shown in FIG. 15. DNA shifts resulting fromtransposome complex formation were analyzed using agarose gelelectrophoresis in 2% agarose gel containing 87 μg/ml BSA and 87 μg/mlheparin in 1×TBE buffer. DNA was stained with ethidium bromide. Theresults show that strong complexes formed only when MuA transposase wascomplexed with control (native) transposon. Weaker complexes were formedwith transposons No. 5, 10, 11. These data correlate well with resultsin FIG. 13. With transposon No. 9 there was no apparent complexformation, indicating that MuA does not bind to this particular DNAsequence. This explains absence of fragmentation using this transposomecomplex shown in FIG. 13.

Experimental results presented in FIGS. 13 and 14 clearly indicate thatdecreased fragmentation level (efficiency) obtained with modifiedtransposons is a consequence of the reduced affinity of MuA transposasetowards the modified transposon ends. Thus, the level of fragmentation,and the average length of the fragmentation products, can be changedeither by varying the sequence of the native transposon end or byvarying the amounts of genomic DNA and preassembled transposome complexcomprising modified transposon ends.

TABLE 3 Double-stranded transposon sequences. Transposon variantDouble-stranded transposon sequence Control5′-GTTTTCGCATTTATCGTGAAACGCTTTCGCGT TTTTCGTGCGTCAGTTCA3′-CAAAAGCGTAAATAGCACTTTGCGAAAGCGCA AAAAGCACGCAGTCAAGTCGT No. 55′-GTCATTACATTGATCGTGAAAGGCTTTCGCGT TGTTCGAGCGCCGCTTTA3′-CAGTAATGTAACTAGCACTTTCCGAAAGCGCA ACAAGCTCGCGGCGAAATCGT No. 95′-GTTCTCGCATCTATCGTGAAAAGCTTCTCCGT TCTTCGGCCGCCGCTTCA3′-CAAGAGCGTAGATAGCACTTTTCGAAGAGGCA AGAAGCCGGCGGCGAAGTCGT No. 105′-GTGCTTTCATTTATCGGGAAACGCTGTCGCGT TGTTCGTGCGCAGCTTTA3′-CACGAAAGTAAATAGCCCTTTGCGACAGCGCA ACAAGCACGCGTCGAAATCGT No. 115′-GTTTGTGCATATATCGTAAAAAGCACTCGCGT ATTTCGTGCGCCGCTTAA3′-CAAACACGTATATAGCATTTTTCGTGAGCGCA TAAAGCACGCGGCGAATTCGT

Example 7 The Use of Tn5 Transpososomes Containing Nicked or GappedTransposon Ends as a Controlled DNA Fragmentation Tool EnablingGeneration of DNA Fragments with Predefined Average Length

The hypervariable Tn5 transposase gene is synthesized, cloned, andoverexpressed in E. coli. Tn5 transposase protein is purified andsynaptic complexes are made with relevant transposon ends, intact orharboring an abasic site, nick, or gap in several locations where it wasshown that such a modified sequence does not interfere with transposasebinding to its recognition sequence. Such transposome complexes areevaluated for their ability to shear DNA in a controlled manner.

The embodiments shown and described in the specification are onlyspecific embodiments of inventors who are skilled in the art and are notlimiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims. Thereferences cited are expressly incorporated by reference herein in theirentirety.

Example 8 The Use of MuA Transposase in Combination with Pre-NickedTransposon Ends for DNA Fragmentation, Adapter Addition andAmplification Reaction in Single Tube

DNA fragments generated using MuA transpososome containing Cut key4(34-16) and Non-cut key4 (42-11) pre-nicked transposon ends and afterfragmentation in same tube produced AA-PCR. This was demonstrated bycomparing the outcome of PCR of E. coli gDNA fragmented with the MuAtranspososome containing the stated nicked transposon end and the MuAtranspososome containing the standard full-length transposon end.

Transposon ends (final concentration 60 μM) were prepared by annealingequimolar quantities of primers Cut-key4 (1Nick34-16), Cut-key4(2Nick34-16) and Non-cut-key4 (1Nick42-11), Non-cut-key4 (2Nick42-11) orCut-key4 (No_nick) and Non-cut-key4 in 10 mM Tris-HCl (pH 8.0), 1 mMEDTA, 50 mM NaCl, following the conditions described in Materials andMethods.

MuA transposomes were formed in complex assembly buffer with extra DMSO.The final concentration of transposon end was 8 μM and for MuAtransposase 1.65 g/l in the complex assembly reaction. After one hourincubation at 30° C., the complex assembly mix was diluted with dilutionbuffer. Final diluted MuA transposome complex concentration was about0.48 g/l. MuA transposome complex was stored at −70° C. for at least 16hours before use.

E. coli gDNA was fragmented in parallel with MuA transpososomecontaining either 34-16/42-11 pre-nicked transposon end or a standardfull length transposon end. Reactions were performed using 100 ng E.coli DNA in AA-PCR reaction buffer. Immediately after adding thetransposome mix (1.5 μl to final reaction volume 30 μl), vortexing and ashort spin-down, the tube was incubated at 30° C. for five minutes.

Fragmented E. coli gDNAs served as templates for PCR amplification withPhusion Hot Start II High-Fidelity DNA polymerase (Thermo Scientific)and 4 primers: A and P1-Ion platform specific adaptors with transposonend sequences, A′ and P1′-PCR amplification primers.

A 5′-CCATCTCATCCCTGCGTGTCTTCGTGCGTCAGTTCA-3′, P15′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATTT CGTGCGTCAGTTCA-3′, A′5′-CCATCTCATCCCTGCGTGTC-3′, P1′ 5′-CCACTACGCCTCCGCTTTCCTCTCTATG-3′.

Reactions were carried out using 30 μl of fragmented DNA (in finalvolume of 50 μl) in 10 mM Tris-HCl, pH 8.8, 110 mM KCl, 1.5 mM MgCl₂,0.1% (w/v), Triton X-100, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 200 μMdGTP using the following cycling conditions: 66° C. 3′, 1×98° C. 30″;9×98° C. 10″, 60° C. 50″, 72° C. 10″; 1×72° C. 1′.

Amplified DNA was purified using the GeneJET NGS Cleanup kit (ThermoFisher Scientific) system. Amplified DNA was transferred into a 1.5 mltube. Then 250 μl of thoroughly mixed GeneJET NGS Cleanup kit (ThermoFisher Scientific) biding buffer and 50 μl of 96% ethanol was added intotube with sample and vortexed. After a short spin, solution wastransferred to the purification column preassembled with a collectiontube and centrifuged for 30 s at 10 000×g. Flow-through was discarded.200 μl pre-wash buffer added to the purification column preassembledwith a collection tube and centrifuged for 30 s at 10 000×g.Flow-through was discarded. 700 μl wash (supplemented with ethanol)buffer added to the purification column preassembled with a collectiontube and centrifuged for 30 s at 10 000×g. Flow-through was discarded.Wash step repeated one more time again. Empty column spined at 14 000 gfor 2 min. to completely remove residual wash buffer. Purificationcolumn was transferred into clean 1.5 mL microcentrifuge tube. 20 μl H₂Owas added to the center of the purification column and centrifuged at 14000×g for 1 min. Supernatants containing the eluted DNA was collected.

DNA samples after PCR amplification were analyzed using the Agilent 2100Bioanalyzer (Agilent Biotechnologies) and the Agilent High SensitivityDNA Kit (Agilent Biotechnologies). Before analysis, fragmented DNA wasdiluted with nuclease free water by a factor of 2, while amplifiedsamples by a factor of 4.

The results are shown in FIG. 16. They revealed that fragmentationreaction and AA-PCR could be combined in the same tube withoutpurification after fragmentation. DNA samples fragmented with MuAtranspososome containing the transposon ends having a nick after 16^(th)position (from the 3′ end) of Cut-key4 oligonucleotide and a nick after11^(th) position (from the 5′ end) of Non-cut-key4 oligonucleotideresulted in reduced amount of DNA fragments with protein-DNA complexesafter fragmentation that are unwanted then producing more than onereaction in the same tube, thus significantly improving the quality ofPCR-amplified DNA library.

1-62. (canceled)
 63. An in vitro method for fragmenting DNA, comprising:a) forming a plurality of transposome complexes by contacting in asingle reaction mixture (i) a plurality of transposases with (ii) aplurality of polynucleotides containing a first transposon end sequence,wherein the first transposon end sequence is capable of binding to atransposase from the plurality of transposases and wherein the firsttransposon end sequence contains at least one nick, gap, apurinic siteor apyrimidinic site, and (iii) a plurality of polynucleotidescontaining a second transposon end sequence, wherein the secondtransposon end sequence is capable of binding to a transposase from theplurality of transposases and wherein the second transposon end sequencecontains at least one nick, gap, apurinic site or apyrimidinic site; andb) contacting the plurality of transposome complexes with a plurality oftarget DNA molecules; and c) producing at least one fragmented DNAmolecule having a first end joined to the first transposon end sequenceand a second end joined to the second transposon end sequence, bytransposing the first and the second transposon end sequences into thetarget DNA molecules and fragmenting the target DNA, wherein the atleast one fragmented DNA molecule includes the first transposon endsequence having at least one nick, gap, apurinic site or apyrimidinicsite, and a second end having at least one nick, gap, apurinic site orapyrimidinic site.
 64. The method of claim 63 wherein the first andsecond transposon ends are selected from a Mu transposon end, a Mos1transposon end, a Vibrio harvey transposon end, and a Tn5 transposonend.
 65. The method of claim 63 wherein the first and second transposaseare selected from a MuA transposase, a Mos1 transposase, a Vibrio harveytransposase, and a Tn5 transposase.
 66. The method of claim 63 furthercomprising the step of contacting the target DNA fragments comprisingthe transposon end at the 5′ ends with DNA polymerase having 5′-3′exonuclease or strand displacement activity, resulting in fullydouble-stranded DNA from the target DNA fragments.
 67. The method ofclaim 63, further comprising: amplifying the at least one fragmented DNAmolecule to produce amplified fragmented DNA molecules.
 68. The methodof claim 67, further comprising: denaturing the amplified target DNA toproduce a plurality of single-stranded fragmented DNA.
 69. The method ofclaim 68, further comprising: immobilizing the plurality ofsingle-stranded fragmented DNA to a support.
 70. The method of claim 69,further comprising: sequencing the plurality of single-strandedfragmented DNA which is immobilized to the support with a massivelyparallel sequencing reaction.
 71. The method of claim 70, wherein themassively parallel sequencing reaction comprises providing a surfacehaving an array of a plurality of reaction sites, and the reaction siteis operatively linked to a sensor.
 72. The method of claim 71, whereinthe sensor detects at least one byproduct or cleavage product of anucleotide incorporation reaction which is selected from a groupconsisting of hydrogen ions, protons and pyrophosphate groups.
 73. Themethod of claim 71, wherein the sensor comprises an ISFET.
 74. Themethod of claim 63, wherein the first transposon end sequence comprisesa double-stranded nucleic acid having a first attacking strand and afirst non-attacking strand.
 75. The method of claim 74, wherein thefirst attacking strand includes a first nick or a first gap.
 76. Themethod of claim 74, wherein the first non-attacking strand includes afirst nick or a first gap.
 77. The method of claim 74, wherein the nickor gap in the first attacking strand is located after the sixth, eighth,tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventhnucleotide from the 3′ end of the first attacking strand.
 78. The methodof claim 63, wherein the second transposon end sequence comprises adouble-stranded nucleic acid having a second attacking strand and asecond non-attacking strand.
 79. The method of claim 78, wherein thesecond attacking strand includes a second nick or a second gap.
 80. Themethod of claim 78, wherein the second non-attacking strand includes asecond nick or second gap.
 81. The method of claim 78, wherein the nickin the second attacking strand is located after the sixth, eighth,tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventhnucleotide from the 3′ end of the second attacking strand.
 82. Themethod of claim 63, wherein the average length of the fragmented DNAmolecules in the plurality can be controlled by (i) varying the amountof transpososome complexes which is contacted with the plurality oftarget DNA, (ii) varying the amount of target DNA which is contactedwith the transpososome complexes, (iii) varying the amount of time ofthe transposition reaction, or (iv) varying the location of the nick orgap on the transposon end sequence.